REGULATION OF SONIC HEDGEHOG ON VASCULAR ENDOTHELIAL GROWTH FACTOR, BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND SECRETION IN BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CAI Jiaqin ¹ , HUANG Yizhou ² , CHEN Xiaohe ³ , XIE Honglei ² , HUANG Yongcan ³ , DENG Li. ³

1Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu Sichuan, 610041, P.R.China;;
2State Key Laboratory of Biotherapy, 3Regenerative Medicine Research Center, Division of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University.;

Corresponding author：

Keywords：

Sonic hedgehog; Bone marrow mesenchymal stem cells; Ischemia related disease; Bone repair; Vascular endothelial growth factor; Basic fibroblast growth factor; Rat

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【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at 【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at

Citation： CAI Jiaqin,HUANG Yizhou,CHEN Xiaohe,XIE Honglei,HUANG Yongcan,DENG Li.. REGULATION OF SONIC HEDGEHOG ON VASCULAR ENDOTHELIAL GROWTH FACTOR, BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND SECRETION IN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(1): 112-116. doi: Copy

1.	Med, 2007, 17(3): 77-83.
2.	signaling. Nat Med, 2005, 11(11): 1197-1204.
3.	of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001, 32(4): 1005-1011.
4.	hedgehog pathway in response to skeletal muscle ischemia. Circulation, 2003, 108(4): 479-485.
5.	neuropathy. Arterioscler Thromb Vasc Biol, 2004, 24(11): 2102-2107.
6.	progenitor cell-mediated microvascular remodeling. Circulation, 2006, 113(20): 2413-2424.
7.	proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro. J Orthop Sci, 2006, 11(5): 491-496.
8.	stem cells improves cardiac function in rats with acute myocardial.
9.	infarction through angiogenesis and myogenesis. Am J Physiol Heart Circ Physiol, 2004, 287(6): 2670-2676.
10.	differentiation and increases pthrp mRNA expression and ipthrp secretion. Bone, 2003, 32(6): 611-620.
11.	and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells. Tissue Eng Part A, 2010, 16(4): 1235-1246.
12.	Cohen MM Jr. The hedgehog signaling network. Am J Med Genet A, 2003, 123A(1): 5-28.
13.	Pola R, Ling L, Silver M, et al. The morphogen sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med, 2001, 7(6): 706-711.
14.	Byrd N, Grabel L. Hedgehog signaling in murine vasculogenesis and angiogenesis. Trends Cardiovasc Med, 2004, 14(8): 308-313.
15.	Lavine KJ, Ornitz DM. Rebuilding the coronary vasculature: hedgehog as a new candidate for pharmacologic revascularization. Trends Cardiovasc.
16.	Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic.
17.	Lavine KJ, Kovacs A, Ornitz DM. Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice. J Clin Invest, 2008, 118(7): 2404-2414.
18.	蔡加琴, 邓力. Hedgehog信号通路对MSCs的调控. 中国修复重建外科杂志, 2010, 24(8): 993-996.
19.	Donahue JK. Gene therapy, angiogenesis, Sonic Hedgehog: Sonic the Hedgehog to the rescue? Gene Ther, 2006, 13(13): 998-999.
20.	Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration.
21.	Tang J, Xie Q, Pan G, et al. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg, 2006, 30(2): 353-361.
22.	Al-Khaldi A, Al-Sabti H, Galipeau J, et al. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. Ann Thorac Surg, 2003, 75(1): 204-209.
23.	Zhou Y, Guan XX, Zhu ZL, et al. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells. Br J Pharmacol, 2010, 161(7): 1542-1552.
24.	金惠铭, 李先涛. 血管新生的调控. 中国微循环, 2001, 5(2): 85-88.
25.	Pola R, Ling L, Aprahamian TR, et al. Postnatal recapitulation of embryonic.
26.	Kusano KF, Allendoerfer KL, Munger W, et al. Sonic hedgehog induces arteriogenesis in diabetic vasa nervorum and restores function in diabetic.
27.	Asai J, Takenaka H, Kusano KF, et al. Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial.
28.	Warzecha J, Göttig S, Brüning C, et al. Sonic hedgehog protein promotes.
29.	Nagaya N, Fujii T, Iwase T, et al. Intravenous administration of mesenchymal.
30.	Jemtland R, Divieti P, Lee K, et al. Hedgehog promotes primary osteoblast.
31.	Yuasa T, Kataoka H, Kinto N, et al. Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2. J Cell Physiol, 2002, 193(2): 225-232.
32.	Dohle E, Fuchs S, Marlen K, et al. Sonic hedgehog promotes angiogenesis.
33.	Suzuki T, Miyamoto T, Fujita N, et al. Osteoblast-specific An gio poi etin1 overexpression increases bone mass. Biochem Biophys Res Commun, 2007, 362(4): 1019-1025.

1. Med, 2007, 17(3): 77-83.
2. signaling. Nat Med, 2005, 11(11): 1197-1204.
3. of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001, 32(4): 1005-1011.
4. hedgehog pathway in response to skeletal muscle ischemia. Circulation, 2003, 108(4): 479-485.
5. neuropathy. Arterioscler Thromb Vasc Biol, 2004, 24(11): 2102-2107.
6. progenitor cell-mediated microvascular remodeling. Circulation, 2006, 113(20): 2413-2424.
7. proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro. J Orthop Sci, 2006, 11(5): 491-496.
8. stem cells improves cardiac function in rats with acute myocardial.
9. infarction through angiogenesis and myogenesis. Am J Physiol Heart Circ Physiol, 2004, 287(6): 2670-2676.
10. differentiation and increases pthrp mRNA expression and ipthrp secretion. Bone, 2003, 32(6): 611-620.
11. and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells. Tissue Eng Part A, 2010, 16(4): 1235-1246.
12. Cohen MM Jr. The hedgehog signaling network. Am J Med Genet A, 2003, 123A(1): 5-28.
13. Pola R, Ling L, Silver M, et al. The morphogen sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med, 2001, 7(6): 706-711.
14. Byrd N, Grabel L. Hedgehog signaling in murine vasculogenesis and angiogenesis. Trends Cardiovasc Med, 2004, 14(8): 308-313.
15. Lavine KJ, Ornitz DM. Rebuilding the coronary vasculature: hedgehog as a new candidate for pharmacologic revascularization. Trends Cardiovasc.
16. Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic.
17. Lavine KJ, Kovacs A, Ornitz DM. Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice. J Clin Invest, 2008, 118(7): 2404-2414.
18. 蔡加琴, 邓力. Hedgehog信号通路对MSCs的调控. 中国修复重建外科杂志, 2010, 24(8): 993-996.
19. Donahue JK. Gene therapy, angiogenesis, Sonic Hedgehog: Sonic the Hedgehog to the rescue? Gene Ther, 2006, 13(13): 998-999.
20. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration.
21. Tang J, Xie Q, Pan G, et al. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg, 2006, 30(2): 353-361.
22. Al-Khaldi A, Al-Sabti H, Galipeau J, et al. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. Ann Thorac Surg, 2003, 75(1): 204-209.
23. Zhou Y, Guan XX, Zhu ZL, et al. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells. Br J Pharmacol, 2010, 161(7): 1542-1552.
24. 金惠铭, 李先涛. 血管新生的调控. 中国微循环, 2001, 5(2): 85-88.
25. Pola R, Ling L, Aprahamian TR, et al. Postnatal recapitulation of embryonic.
26. Kusano KF, Allendoerfer KL, Munger W, et al. Sonic hedgehog induces arteriogenesis in diabetic vasa nervorum and restores function in diabetic.
27. Asai J, Takenaka H, Kusano KF, et al. Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial.
28. Warzecha J, Göttig S, Brüning C, et al. Sonic hedgehog protein promotes.
29. Nagaya N, Fujii T, Iwase T, et al. Intravenous administration of mesenchymal.
30. Jemtland R, Divieti P, Lee K, et al. Hedgehog promotes primary osteoblast.
31. Yuasa T, Kataoka H, Kinto N, et al. Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2. J Cell Physiol, 2002, 193(2): 225-232.
32. Dohle E, Fuchs S, Marlen K, et al. Sonic hedgehog promotes angiogenesis.
33. Suzuki T, Miyamoto T, Fujita N, et al. Osteoblast-specific An gio poi etin1 overexpression increases bone mass. Biochem Biophys Res Commun, 2007, 362(4): 1019-1025.

Chinese Journal of Reparative and Reconstructive Surgery

REGULATION OF SONIC HEDGEHOG ON VASCULAR ENDOTHELIAL GROWTH FACTOR, BASIC FIBROBLAST GROWTH FACTOR EXPRESSION AND SECRETION IN BONE MARROW MESENCHYMAL STEM CELLS

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