BACKGROUND
angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is a closely regulated sequence of events beginning with the degradation of the basement membrane by activated endothelial cells .
these then migrate and proliferate, form endothelial sprouts and develop capillary tubes and a new basement membrane.
the key events of angiogenesis therefore involve ec proliferation, migration, tube formation and differentiation into capillaries  <cit> .
angiogenesis is associated with normal physiological  and pathological processes   <cit> .
angiogenesis is regulated by a balance between endogenous, soluble pro-angiogenic factors   <cit> , fibroblast growth factor- <dig>   <cit> , epidermal growth factor  and angiopoietins, and anti-angiogenic factors   <cit> .
growth factors exert their effect through binding to their cognate receptor; for example the kinase insert domain-containing receptor  and tie- <dig> receptors   <cit> .
fgfs exert their effect by binding to high affinity fgf-receptors  on the cell surface.
in vitro, ecs express fgfr- <dig> and in some cases fgfr- <dig> but not fgfr- <dig> or - <dig>  <cit> .
because de-regulated angiogenesis is associated with disease progression, especially tumour development, inhibition of neo-vessel growth has become a target in drug development.
natural compounds from medicinal plants display diverse pharmacological activities and have advantages over synthetic drugs, such as smoother action, better tolerance and fewer allergic reactions  <cit> .
for example anti-angiogenic plant derived natural products such as genistein  <cit> , isoliquitrin  <cit> , ginsenoside <cit>  and torilin  <cit>  have potent effects on ec proliferation or tube formation.
stilbene glycosides are natural products isolated from the medicinal plant euphobia chiradenia and in preliminary screening were shown to be pla <dig> inhibitors, have anti-inflammatory properties and inhibit wound healing although the mechanism of action was not investigated  <cit> .
based on these results we speculated that stilbene glycosides may be anti-angiogenic and tested the efficacy of two of these compounds, trans-4',5'-dihydroxy-3-methoxystilbene-5-o-{α-l-rhamnopyranosyl-- -β-d-glucopyranoside   against large and small vessel-derived ec in a range of in vitro and in vivo angiogenic assays.
RESULTS
toxicity
compounds  <dig> and  <dig> had no significant cytotoxic effect on bovine aortic endothelial cells  and human dermal microvascular endothelial cells  over the concentration range used whereas staurosporine  showed significant cytotoxicity.
representative data for baec are shown in figure  <dig>
the effect of compounds  <dig> and  <dig> on growth factor-induced proliferation
compounds  <dig> and  <dig> at concentrations of  <dig> – <dig>  μm had no significant effect on baec and hdmec growth in the absence of growth factors .
over a range of concentrations compound  <dig> inhibited fgf-2-induced baec and hdmec proliferation in a dose dependent manner with ic <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm respectively .
compound  <dig> was a less effective inhibitor and inhibited hdmec  but had no activity against large vessel baec .
the compounds had little effect on vascular endothelial growth factor  or epidermal growth factor  stimulated proliferation .
ns = not significant, overall inhibition is less than 50% and therefore ic <dig> values cannot be calculated.
nd = not determined
figures are the ic <dig> values ± sd  and are the mean of at least three determinations.
the effect of compounds  <dig> and  <dig> on endothelial cell migration during wound healing
two- and three-dimensional cell migration assays were used to determine whether compounds  <dig> and  <dig> had an effect on ec cell migration.
in a wound healing model, cell monolayers were wounded and exposed to compounds  <dig> or  <dig> with and without fgf- <dig>  fgf- <dig> at a final concentration of  <dig> ng/ml induced significant migration into the denuded area  and this was inhibited by compound  <dig> with an ic <dig> value of  <dig>  ±  <dig>  μm .
compound  <dig> was ineffective.
representative photomicrographs are shown .
the chemotactic effect of compounds  <dig> and  <dig> was measured in the three-dimensional boyden chamber assay.
baec and hdmec stimulated by fgf- <dig>  showed a significant increase in migration .
compound  <dig> inhibited migration of both cell types in a dose-dependent manner with ic <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm respectively .
compound  <dig> was ineffective with ic <dig> values in excess of  <dig> μm.
the effect of compounds  <dig> and  <dig> on endothelial tube formation
in the presence of fgf- <dig>  there was an increase in baec and hdmec differentiation into capillary-like structures .
this process was inhibited by compound  <dig> in a dose dependent manner with ic <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm  for baec and hdmec respectively.
figure  <dig> shows representative results for baec.
compound  <dig> had no inhibitory activity.
the effect of compounds  <dig> and  <dig> on the regression of established tubular networks formed from baec was also investigated.
compound  <dig> had an ic <dig> value of  <dig>  ±  <dig>  and compound  <dig> of  <dig>  ±  <dig>  μm respectively .
the effect of compounds  <dig> and  <dig> on endothelial invasion in matrigel
the effect of the test compounds on cell invasion was investigated using a transwell boyden chamber system coated with reconstituted growth factor-depleted matrigel.
baec and hdmec treated with fgf- <dig> showed a  <dig>  and  <dig>  fold increase in migration into a second layer of matrigel respectively .
this process was inhibited by compound  <dig> with ic <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm  and compound  <dig> with ic <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm  for baec and hdmec respectively.
representative photomicrographs for the effect of compound  <dig> are shown .
binding studies with fgf- <dig> receptors
the anti-angiogenic effect of compounds  <dig> and  <dig> may occur by either competing with fgf- <dig> for its receptors or by altering receptor binding.
if compound  <dig> and  <dig> were added with fgf- <dig> to fgfr- <dig> or fgfr- <dig> no significant inhibitory effect was observed.
however, if the compounds were pre-incubated with fgf- <dig> a significant reduction of fgf- <dig> binding for compound  <dig>  was observed .
compound  <dig> had no inhibitory effect and neither compounds affected binding to the vegf receptor .
the inhibition of erk1/ <dig> phosphorylation by compounds  <dig> and 2
fgf- <dig> induced cell proliferation, migration and differentiation is mediated through receptor binding and associated intracellular signal pathways involving erk1/ <dig>  <cit> .
fgf- <dig> addition caused a 50% increase in erk1/ <dig> phosphorylation compared to the control.
this was inhibited by compound  <dig> in a dose-dependent manner .
compound  <dig> was ineffective.
inhibition of angiogenesis in the chick chorioallantoic membrane  assay
since the in vitro assays described above suggest inhibition of several steps of angiogenesis we next studied the interaction of compounds  <dig> and  <dig> with fgf- <dig> in an in vivo system, the cam assay.
there was no evidence of angiogenesis or inflammation with the control, methylcellulose, used for the addition of the test compounds .
after exposure to fgf- <dig>  there was a significant increase in the formation of new blood vessels growing radially towards the stimulus  .
the determination of the degree of angiogenesis  is described below in the materials and methods section.
after  <dig> days exposure to the test compounds  there was a 100% reduction in fgf-2-induced angiogenesis in the case of compound  <dig>  and 90% reduction with compound  <dig>  with a notable inhibition in the formation of normal cam blood vessels.
the test compounds alone were not inflammatory or angiogenic .
hydrophobicity of  <dig> and 2
the hydrophobicity descriptor  for compounds  <dig> and  <dig> were - <dig>  and  <dig>  respectively indicating that compound  <dig> is approximately an order of magnitude more water soluble than  <dig> but both compounds are very water soluble.
the effect of compounds  <dig> and  <dig> on egf and vegf-induced angiogenesis
compounds  <dig> or  <dig> had no significant inhibitory effect on egf or vegf-induced baec- or hdmec growth or differentiation as measured in the angiogenesis assays described above .
discussion and
CONCLUSIONS
we have demonstrated the ability of compound  <dig>  a plant-derived stilbene glycoside to inhibit multiple stages of angiogenesis in in vivo and in vitro assays with ic <dig> values in the range of  <dig>  ±  <dig>  to  <dig>  ±  <dig>  μm.
compound  <dig> was equally effective against small and large vessel-derived ecs but compound  <dig> was largely ineffective.
compound  <dig> which contains an additional bulky l-rhamnose residue and is approximately  <dig> times more water soluble than compound  <dig> was the more effective inhibitor.
since the compounds show approximately 90% structural homology, activity appears to be inversely related to hydrophobicity.
we have shown using other plant-derived natural products that increased polarity correlates with effectiveness of growth factor inhibition.
for example, the ability of sesterterpenes to inhibit fgf-2-induced angiogenesis  <cit>  and cheiradone to inhibit vegf  <cit>  increased with polarity.
stilbene glycosides and their derivatives are naturally occurring phytoalexins, widely distributed and with diverse activities.
for example resveratrol, a stilbene glucoside inhibited the differentiation of human umbilical vein ecs into capillary-like structures at concentrations of  <dig> – <dig> mm  <cit>  and induced apoptosis in colon cancer cells  <cit> .
angiogenesis is an invasive process that requires degradation of the basement membrane, cell migration and removal of obstructing matrix proteins to allow space for the formation of the vessel lumen  <cit> .
matrix metalloproteinases , in particular mmp- <dig> and - <dig> are the principal mediators of these events  <cit> .
stilbene glycosides are potent anti-oxidants with ic <dig> values in the μm range  <cit>  and may suppress mmp expression, and therefore angiogenesis by decreasing oxidative stress, a known inducer of mmp- <dig>  this may indirectly regulate angiogenesis  <cit> .
however, we were able to show that the major effect of compound  <dig> was in reducing fgf- <dig> induced angiogenesis.
in the absence of growth factors, stilbene glycosides had little effect on ec proliferation and migration.
however, compound  <dig> bound to fgf- <dig> reducing its binding to its cognate receptors  with ic <dig> values of  <dig>  to  <dig>  μm respectively.
the effect was specific with no inhibition of egf or vegf activity and compound  <dig> did not bind in vitro to the vegf receptor.
angiogenesis is regulated by a balance between pro- and anti-angiogenic regulators.
if the pro-angiogenic stimulators predominate ecs switch to an angiogenic phenotype.
it appears that stilbene glycosides regulate angiogenesis by decreasing the activity of pro-angiogenic fgf- <dig>  in addition compounds  <dig> and  <dig> showed activity in the in vivo cam assay.
the suppression of fgf- <dig> induced vascular networks was accomplished without inflammation or embryo death.
in vitro, compound  <dig> reduced the formation of vascular networks by baecs and huvecs on matrigel beds and caused regression of established networks.
compound  <dig> also reduced fgf- <dig> induced proliferation and migration of ecs.
it is possible therefore, that compound  <dig> may have a role in reducing excessive angiogenesis.
activation of signal transduction pathways follows binding of growth factors to receptor tyrosine kinases and in the case of fgf- <dig> involves phosphorylation of erk- <dig> and - <dig>  culminating in the activation of the transcription factor c-jun and initiation of the early events of angiogenesis  <cit> .
we were able to show that compound  <dig> reduced levels of phospho-erk- <dig> and - <dig> in fgf- <dig> stimulated ecs in a dose-response manner.
the few known natural product inhibitors of fgf- <dig> include two sesterterpenes, leucosesterterpene  <cit>  and torilin  <cit>  and the aporphine alkaloid sinomenine  <cit> .
both torilin and sinomenine show potential as anti-tumour agents, the latter is active against synovial carcinoma  <cit> .
since compound  <dig> shows anti-angiogenic activity with similar ic <dig> values to sinomenine there is a possibility that it may have potential as an anti-tumour agent.

 angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is a closely regulated sequence of events beginning with the degradation of the basement membrane by activated endothelial cells. these then migrate and proliferate, form endothelial sprouts and develop capillary tubes and a new basement membrane. the key events of angiogenesis therefore involve ec proliferation, migration, tube formation and differentiation into capillaries. angiogenesis is associated with normal physiological and pathological processes. angiogenesis is regulated by a balance between endogenous, soluble pro-angiogenic factors, fibroblast growth factor-, epidermal growth factor and angiopoietins, and anti-angiogenic factors. growth factors exert their effect through binding to their cognate receptor; for example the kinase insert domain-containing receptor and tie- receptors. fgfs exert their effect by binding to high affinity fgf-receptors on the cell surface. in vitro, ecs express fgfr- and in some cases fgfr- but not fgfr- or -. because de-regulated angiogenesis is associated with disease progression, especially tumour development, inhibition of neo-vessel growth has become a target in drug development. natural compounds from medicinal plants display diverse pharmacological activities and have advantages over synthetic drugs, such as smoother action, better tolerance and fewer allergic reactions. for example anti-angiogenic plant derived natural products such as genistein, isoliquitrin, ginsenoside and torilin have potent effects on ec proliferation or tube formation. stilbene glycosides are natural products isolated from the medicinal plant euphobia chiradenia and in preliminary screening were shown to be pla inhibitors, have anti-inflammatory properties and inhibit wound healing although the mechanism of action was not investigated. based on these results we speculated that stilbene glycosides may be anti-angiogenic and tested the efficacy of two of these compounds, trans-4',5'-dihydroxy-3-methoxystilbene-5-o-{α-l-rhamnopyranosyl-- -β-d-glucopyranoside against large and small vessel-derived ec in a range of in vitro and in vivo angiogenic assays.  toxicity compounds and had no significant cytotoxic effect on bovine aortic endothelial cells and human dermal microvascular endothelial cells over the concentration range used whereas staurosporine showed significant cytotoxicity. representative data for baec are shown in figure the effect of compounds and on growth factor-induced proliferation compounds and at concentrations of – μm had no significant effect on baec and hdmec growth in the absence of growth factors. over a range of concentrations compound inhibited fgf-2-induced baec and hdmec proliferation in a dose dependent manner with ic values of ± and ± μm respectively. compound was a less effective inhibitor and inhibited hdmec but had no activity against large vessel baec. the compounds had little effect on vascular endothelial growth factor or epidermal growth factor stimulated proliferation. ns = not significant, overall inhibition is less than 50% and therefore ic values cannot be calculated. nd = not determined figures are the ic values ± sd and are the mean of at least three determinations. the effect of compounds and on endothelial cell migration during wound healing two- and three-dimensional cell migration assays were used to determine whether compounds and had an effect on ec cell migration. in a wound healing model, cell monolayers were wounded and exposed to compounds or with and without fgf- fgf- at a final concentration of ng/ml induced significant migration into the denuded area and this was inhibited by compound with an ic value of ± μm. compound was ineffective. representative photomicrographs are shown. the chemotactic effect of compounds and was measured in the three-dimensional boyden chamber assay. baec and hdmec stimulated by fgf- showed a significant increase in migration. compound inhibited migration of both cell types in a dose-dependent manner with ic values of ± and ± μm respectively. compound was ineffective with ic values in excess of μm. the effect of compounds and on endothelial tube formation in the presence of fgf- there was an increase in baec and hdmec differentiation into capillary-like structures. this process was inhibited by compound in a dose dependent manner with ic values of ± and ± μm for baec and hdmec respectively. figure shows representative results for baec. compound had no inhibitory activity. the effect of compounds and on the regression of established tubular networks formed from baec was also investigated. compound had an ic value of ± and compound of ± μm respectively. the effect of compounds and on endothelial invasion in matrigel the effect of the test compounds on cell invasion was investigated using a transwell boyden chamber system coated with reconstituted growth factor-depleted matrigel. baec and hdmec treated with fgf- showed a and fold increase in migration into a second layer of matrigel respectively. this process was inhibited by compound with ic values of ± and ± μm and compound with ic values of ± and ± μm for baec and hdmec respectively. representative photomicrographs for the effect of compound are shown. binding studies with fgf- receptors the anti-angiogenic effect of compounds and may occur by either competing with fgf- for its receptors or by altering receptor binding. if compound and were added with fgf- to fgfr- or fgfr- no significant inhibitory effect was observed. however, if the compounds were pre-incubated with fgf- a significant reduction of fgf- binding for compound was observed. compound had no inhibitory effect and neither compounds affected binding to the vegf receptor. the inhibition of erk1/ phosphorylation by compounds and 2 fgf- induced cell proliferation, migration and differentiation is mediated through receptor binding and associated intracellular signal pathways involving erk1/. fgf- addition caused a 50% increase in erk1/ phosphorylation compared to the control. this was inhibited by compound in a dose-dependent manner. compound was ineffective. inhibition of angiogenesis in the chick chorioallantoic membrane assay since the in vitro assays described above suggest inhibition of several steps of angiogenesis we next studied the interaction of compounds and with fgf- in an in vivo system, the cam assay. there was no evidence of angiogenesis or inflammation with the control, methylcellulose, used for the addition of the test compounds. after exposure to fgf- there was a significant increase in the formation of new blood vessels growing radially towards the stimulus. the determination of the degree of angiogenesis is described below in the materials and methods section. after days exposure to the test compounds there was a 100% reduction in fgf-2-induced angiogenesis in the case of compound and 90% reduction with compound with a notable inhibition in the formation of normal cam blood vessels. the test compounds alone were not inflammatory or angiogenic. hydrophobicity of and 2 the hydrophobicity descriptor for compounds and were - and respectively indicating that compound is approximately an order of magnitude more water soluble than but both compounds are very water soluble. the effect of compounds and on egf and vegf-induced angiogenesis compounds or had no significant inhibitory effect on egf or vegf-induced baec- or hdmec growth or differentiation as measured in the angiogenesis assays described above. discussion and  we have demonstrated the ability of compound a plant-derived stilbene glycoside to inhibit multiple stages of angiogenesis in in vivo and in vitro assays with ic values in the range of ± to ± μm. compound was equally effective against small and large vessel-derived ecs but compound was largely ineffective. compound which contains an additional bulky l-rhamnose residue and is approximately times more water soluble than compound was the more effective inhibitor. since the compounds show approximately 90% structural homology, activity appears to be inversely related to hydrophobicity. we have shown using other plant-derived natural products that increased polarity correlates with effectiveness of growth factor inhibition. for example, the ability of sesterterpenes to inhibit fgf-2-induced angiogenesis and cheiradone to inhibit vegf increased with polarity. stilbene glycosides and their derivatives are naturally occurring phytoalexins, widely distributed and with diverse activities. for example resveratrol, a stilbene glucoside inhibited the differentiation of human umbilical vein ecs into capillary-like structures at concentrations of – mm and induced apoptosis in colon cancer cells. angiogenesis is an invasive process that requires degradation of the basement membrane, cell migration and removal of obstructing matrix proteins to allow space for the formation of the vessel lumen. matrix metalloproteinases, in particular mmp- and - are the principal mediators of these events. stilbene glycosides are potent anti-oxidants with ic values in the μm range and may suppress mmp expression, and therefore angiogenesis by decreasing oxidative stress, a known inducer of mmp- this may indirectly regulate angiogenesis. however, we were able to show that the major effect of compound was in reducing fgf- induced angiogenesis. in the absence of growth factors, stilbene glycosides had little effect on ec proliferation and migration. however, compound bound to fgf- reducing its binding to its cognate receptors with ic values of to μm respectively. the effect was specific with no inhibition of egf or vegf activity and compound did not bind in vitro to the vegf receptor. angiogenesis is regulated by a balance between pro- and anti-angiogenic regulators. if the pro-angiogenic stimulators predominate ecs switch to an angiogenic phenotype. it appears that stilbene glycosides regulate angiogenesis by decreasing the activity of pro-angiogenic fgf- in addition compounds and showed activity in the in vivo cam assay. the suppression of fgf- induced vascular networks was accomplished without inflammation or embryo death. in vitro, compound reduced the formation of vascular networks by baecs and huvecs on matrigel beds and caused regression of established networks. compound also reduced fgf- induced proliferation and migration of ecs. it is possible therefore, that compound may have a role in reducing excessive angiogenesis. activation of signal transduction pathways follows binding of growth factors to receptor tyrosine kinases and in the case of fgf- involves phosphorylation of erk- and - culminating in the activation of the transcription factor c-jun and initiation of the early events of angiogenesis. we were able to show that compound reduced levels of phospho-erk- and - in fgf- stimulated ecs in a dose-response manner. the few known natural product inhibitors of fgf- include two sesterterpenes, leucosesterterpene and torilin and the aporphine alkaloid sinomenine. both torilin and sinomenine show potential as anti-tumour agents, the latter is active against synovial carcinoma. since compound shows anti-angiogenic activity with similar ic values to sinomenine there is a possibility that it may have potential as an anti-tumour agent. 
compound inhibited fgf- induced angiogenesis by binding to its cognate receptors and is an addition to the small number of natural product inhibitors of angiogenesis compound -[α-l-rhamnopyranosyl-}-β-d-glucopyranoside was the more hydrophilic and inhibited fgf-2-induced proliferation, wound healing, invasion in matrigel, tube formation and angiogenesis in large and small vessel-derived endothelial cells and also in the chick chorioallantoic membrane assay. we have investigated the anti-angiogenic potential of two naturally occurring stilbene glycosides isolated from the medicinal plant boswellia papyriferai using large and smallvessel-derived endothelial cells. angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with pathological processes, in particular tumour development, and is a target for the development of new therapies.
 angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with pathological processes, in particular tumour development, and is a target for the development of new therapies. we have investigated the anti-angiogenic potential of two naturally occurring stilbene glycosides isolated from the medicinal plant boswellia papyriferai using large and smallvessel-derived endothelial cells. compound -[α-l-rhamnopyranosyl-}-β-d-glucopyranoside was the more hydrophilic and inhibited fgf-2-induced proliferation, wound healing, invasion in matrigel, tube formation and angiogenesis in large and small vessel-derived endothelial cells and also in the chick chorioallantoic membrane assay. using a binding assay we were able to show compound reduced binding of fgf- to fibroblast growth factor receptors- and - in all cases the concentration of compound which caused 50% inhibition was determined. the effect of compound on egf and vegf-induced proliferation was also investigated.  compound inhibited all stages of fgf- induced angiogenesis with ic values in the range ± – ± μm but did not inhibit egf or vegf-induced angiogenesis. it also inhibited fgf- binding to fgf receptor- and - with ic values of ± and ± μm respectively and with concommotant down-regulation of phosphorylated-erk-1/- expression. compound was an ineffective inhibitor of angiogenesis despite its structural homology to compound  compound inhibited fgf- induced angiogenesis by binding to its cognate receptors and is an addition to the small number of natural product inhibitors of angiogenesis 