GinsenosideRg1 mediates a hypoxiaindependent upr

Authors: KarWah Leung HoiMan Ng Maggie K S Tang Chris C K Wong Ricky N S Wong Alice S T Wong

Publish Date: 2011/10/02

Volume: 14, Issue: 4, Pages: 515-522

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Abstract

Hypoxiainducible factor HIF1 is the key transcription regulator for multiple angiogenic factors and is an appealing target GinsenosideRg1 a nontoxic saponin isolated from the rhizome of Panax ginseng exhibits potent proangiogenic activity and has the potential to be developed as a new angiotherapeutic agent However the mechanisms by which Rg1 promotes angiogenesis are not fully understood Here we show that Rg1 is an effective stimulator of HIF1α under normal cellular oxygen conditions in human umbilical vein endothelial cells HIF1α steadystate mRNA was not affected by Rg1 Rather HIF1α protein synthesis was stimulated by Rg1 This effect was associated with constitutive activation of phosphatidylinositol 3kinase PI3K/Akt and its effector p70 S6 kinase p70S6K but not extracellularsignal regulated kinase 1/2 We further revealed that HIF1α induction triggered the expression of target genes including vascular endothelial growth factor VEGF The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70S6K activities respectively resulted in diminished HIF1α activation and subsequent VEGF expression RNA interferencemediated knockdown of HIF1α suppressed Rg1induced VEGF synthesis and angiogenic tube formation confirming that the effect was HIF1α specific Similarly the angiogenic phenotype could be reversed by inhibition of PI3K/Akt and p70S6K These results define a hypoxiaindependent activation of HIF1α uncovering a novel mechanism for Rg1 that could play a major role in angiogenesis and vascular remodelingInsufficient angiogenesis is a major pathological component of diseases such as chronic wounds and ischemic heart disease Therapeutic angiogenesis aims at solving this problem by stimulating new vessel formation 1 Given its central role in angiogenesis vascular endothelial growth factor VEGF has become a prime target for angiotherapy However clinical trials have not achieved satisfactory results due to aberrant functions of the overexpressed protein Hypoxiainducible factor1 HIF1 which can stimulate the required angiogenic growth factors endogenously has been suggested to provide an advantage over VEGF therapy 2Hypoxiainducible factor is a master regulator of the transcriptional response of angiogenesis 3 HIF1 is a heterodimer consisting of subunits HIF1α and HIF1β 4 HIF1β is constitutively expressed whereas HIF1α is tightly regulated by changes in oxygen regimes 5 6 HIF1α expression is regulated by multiple mechanisms Although it primarily involves protein ubiquitination the accumulation of HIF1α has also been shown to depend on its rate of de novo protein synthesis 7 Depending on the cell types and stimuli different signaling pathways are involved in regulation of HIF1α expression Once activated HIF1 regulates a repertoire of key angiogenic genes including VEGF plateletderived growth factor PDGF and transforming growth factor α TGFα 3 Thus HIF1 represents an appealing drug target Nevertheless targeted therapy in clinical settings in general is not sufficient to treat angiogenic diseases partly due to inadequate delivery strategies 8 Plantbased small molecule activators that exhibit low toxicity and side effects hold promise as new treatment optionsEmerging evidence shows that nonpeptide small molecules such as saponins can modulate angiogenesis with great potential to be developed as angiotherapeutic agents 9 Panax ginseng containing saponins as the major and biological active components is a key herb in traditional Chinese medicine reported to be efficacious in treating diabetes and cardiovascular concerns and its consumption is safe and nontoxic even at high doses in animals and humans 10 A notable saponin isolated from P ginseng is Rg1 11 We have previously shown strong proangiogenic efficacy of Rg1 In particular Rg1 was found to potently promote human umbilical vein endothelial cell HUVEC proliferation chemoinvasion and tube formation in vitro to stimulate neovascularization in vivo and to induce outgrowth of aortic sprout ex vivo 12 Given this potential as a new attractive modality for angiotherapy it is of great interest to characterize the signal transduction pathway whereby Rg1 contributes to angiogenesisWe have used HUVECs based on its ability to undergo in vitro angiogenesis in response to appropriate stimuli but also a standard line for angiogenic screening to provide understanding of the mechanisms of Rg1 Using this model we show for the first time that Rg1 is a potent stimulator of HIF1α under normal oxygen conditions We also provide mechanistic insights suggesting that the activation of the translation signal especially p70S6K through the PI3K/Akt mediated pathway in this process This hypoxiaindependent activation of HIF1α elucidates an important mechanism which may explain the promising effects of Rg1 on angiogenesis and provide a rationale for the development of Rg1 as a new source of small molecule angiomodulatorHuman umbilical vein endothelial cells were obtained from Clonetics San Diego CA and cultured in medium 199 supplemented with 20 fetal bovine serum 20 μg/ml endothelial cell growth supplement 90 U/ml heparin and 1 penicillin–streptomycin in a humidified incubator at 37°C with 5 CO2 The fifth to eighth passages of HUVECs were used in these studies to ensure genetic stability of the culture GinsenosideRg1 is a reference compound purity  98 purchased from the Division of Chinese Material Medica and Natural Products National Institute for the Control of Pharmaceutical and Biological Products Ministry of Public Health China A stock solution of Rg1 50 mM was prepared in sterile double distilled waterCells were lysed in RIPA buffer 150 mM NaCl 50 mM Tris pH 74 2 mM EDTA 02 SDS and 1 Triton X100 Lysates were cleared by centrifugation and protein concentrations were determined using the Bradford method with reagents from BioRad Hercules CA Equal amounts of cell lysates were separated by SDS–PAGE and transferred to a nitrocellulose membrane The blot was then probed with HIF1α and HIF1β BD Transduction Laboratories San Jose CA 1500 VEGF Santa Cruz Biotechnology Santa Cruz CA 11000 phosphoAkt total Akt phosphop70S6K total p70S6K Cell Signaling Danvers MA 11000 and βactin Sigma St Louis MO 11000 followed by reaction with horseradish peroxidaseconjugated secondary antibody The signal was detected using enhanced chemiluminescence Amersham Piscataway NJ

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