Crude Extract of Salvia miltiorrhiza and Salvianolic Acid B Enhance in Vitro Angiogenesis in Murine SVR Endothelial Cell Line

Abstract

Salvia miltiorrhiza (SM) has been used clinically in Asian countries to improve the microcirculation in the human body. Although a pure compound extracted from SM, salvianolic acid B (Sal B), has been reported to be effective against fibrosis and ischemia-reperfusion injury, possibly through its anti-lipid peroxidation action, the effect of SM on angiogenesis remains unclear. It is our interest to investigate the role of SM on the regulation of the angiogenic process. By using the SVR endothelial cell line as an in vitro system, the effects of Sal B on the gene expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, VEGF-R2 were evaluated by morphology, differentiation assay, reverse-transcribed polymerase chain reaction (RT-PCR) and Western blot analysis. The results showed that both the crude extract of SM and the pure compound Sal B had enhancing effects on cell growth and differentiation. The gene expression of matrix metalloproteinase-2 (MMP-2) was up-regulated after Sal B treatment for 2 h, while VEGF and VEGF-R2 gene expression were up-regulated 40 min after Sal B treatment. We conclude that the crude extract of SM and Sal B enhance angiogenic processes on SVR cells through up-regulation of VEGF and VEGF receptors genes.

Key words

Salvia miltiorrhiza - Lamiaceae - salvianolic acid B - angiogenesis - VEGF - endothelial cells

References

• 1 Salcedo R, Wasserman K, Young H A, Grimm M C, Howard O M, Anver M R. et al . Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha.  Am J Pathol. 1999;  154 1125-35
• 2 Giordano F J. Angiogenesis: mechanisms, modulation, and targeted imaging.  J Nucl Cardiol. 1999;  6 664-71
• 3 Hanahan D. Signaling vascular morphogenesis and maintenance.  Science. 1997;  277 48-50
• 4 Shamamian P, Schwartz J D, Pocock B J, Monea S, Whiting D, Marcus S G, et al. Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis.  J Cell Physiol. 2001;  189 197-206
• 5 Freedman S B, Isner J M. Therapeutic angiogenesis for ischemic cardiovascular disease.  J Mol Cell Cardiol. 2001;  33 379-93
• 6 Zhou W, Ruigrok T JC. Protective effect of Danshen during myocardial ischemia and reperfusion: an isolated rat heart study.  Am J Chin Med. 1990;  18 19-24
• 7 Lei X L, Chiou G C. Cardiovascular pharmacology of Panax notoginseng (Burk) F.H. Chen and Salvia miltiorrhiza .  Am J Chin Med. 1986;  14 145-52
• 8 Wu Y J, Hong C Y, Lin S J, Wu P, Shiao M S. Increase of vitamin E content in LDL and reduction of atherosclerosis in cholesterol-fed rabbits by a water-soluble antioxidant-rich fraction of Salvia miltiorrhiza .  Arterioscl Throm Vas. 1998;  18 481-6
• 9 Liu J, Yang C F, Wasser S, Shen H M, Tan C E, Ong C N. Protection of Salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats: dual mechanisms involved.  Life Sci. 2001;  69 309-26
• 10 Nan J X, Park E J, Kang H C, Park P H, Kim J Y, Sohn D H. Anti-fibrotic effects of a hot-water extract from Salvia miltiorrhiza roots on liver fibrosis induced by biliary obstruction in rats.  J Pharm Pharmacol. 2001;  53 197-204
• 11 Liu J, Shen H M, Ong C N. Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma HepG(2) cells.  Cancer Lett. 2000;  153 85-93
• 12 Chen Y H, Du G H, Zhang J T. Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats.  Acta Pharmacol Sin. 2000;  21 463-6
• 13 Patterson C, Runge M S. Therapeutic myocardial angiogenesis via vascular endothelial growth factor gene therapy: moving on down the road.  Circulation. 2000;  102 940-2
• 14 Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois R N. Cyclooxygenase regulates angiogenesis induced by colon cancer cells.  Cell. 1998;  93 705-16
• 15 Nicosia R F, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro .  Lab Inves. 1990;  63 15-22
• 16 Wasser S, Ho J M, Ang H K, Tan C E. Salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats.  J Hepatol. 1998;  29 760-71
• 17 Arbiser J L, Larsson H, Claesson-Welsh L, Bai X, LaMontagne K, Weiss S W. et al . Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR-1 and VEGFR-2 in vivo .  Am J Pathol. 2000;  156 1469-76
• 18 Arbiser J L, Moses M A, Fernandez C A, Ghiso N, Cao Y, Klauber N. et al . Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.  Proc Natl Acad Sci USA. 1997;  94 861-6
• 19 Folkman J, Ingber D. Inhibition of angiogenesis.  Seminars in Cancer Biology. 1992;  3 89-96
• 20 Liu P, Liu N, Xu L, Hu Y, Ji G, Liu C. Direct protection of injured primary cultured hepatocytes of rats treated with carbon tetrachloride by salvianic acid B.  [in Chinese] Chung-Kuo Chung Yao Tsa Chih - China Journal of Chinese Materia Medica. 1997;  22 303-5
• 21 Hung H H, Chen Y L, Lin S J, Yang S P, Shih C C, Shiao M S. et al . A salvianolic acid B-rich fraction of Salvia miltiorrhiza induces neointimal cell apoptosis in rabbit angioplasty model.  Histol Histopathol. 2001;  16 175-83