BACKGROUND
the cytokine transforming growth factor beta  is a member of the tgf-β superfamily consisting of  <dig> members including tgf-βs   <cit> .
tgf-β <dig> is the predominant and more ubiquitous form.
tgf-β ligands signal canonically through type i  and type ii serine/threonine kinase receptors  <cit> , and via the accessory  receptor endoglin in vascular endothelial cells  <cit> .
tgf-β binding leads to phosphorylation of intracellular r-smads  <dig>   <dig>   <dig>   <dig> or  <dig>  <cit> , which then complex with co-smad <dig>  enter the nucleus and associate with transcriptional co-activators or co-repressors to regulate the expression of target genes.
tgf-β plays a dual role in angiogenesis by orchestrating a switch from vascular inhibition to pro-angiogenic activity .
in particular, there is evidence that tgf-β <dig> induced angiogenesis acts with vegf to mediate apoptosis of excessive vascular sprouts and may even be required for initial sprouting from an existing vascular network  <cit> .
the nature of the angiogenic response to tgf-β depends on the balance of alk <dig> versus alk <dig> signaling input, with alk <dig> predominantly promoting sprouting and alk <dig> favoring the resolution/stabilization phase of angiogenesis  <cit> .
thus, tgf-β is either pro- or anti-angiogenic, depending on which tgf-rii/smad pathway is engaged  <cit> .
differences also exist in the kinetics and dose responses of these pathways: alk <dig> mediated signaling is transient and maximal at low concentrations of ligand while alk <dig> mediated signaling is sustained and maximal at higher doses of ligand  <cit> .
increased expression of endoglin led to inhibition of tgf-β/alk <dig> signaling  in a dose dependent fashion  <cit> .
activation of vascular sprouting during angiogenesis entails the specification of endothelial cells into tip and stalk cells.
endothelial tip cells are mainly migratory and polarized with minimal proliferation while stalk cells proliferate throughout sprout establishment and form the nascent vascular lumen cells  <cit> .
the delta-like ligand  <dig> -notch <dig> signaling pathway is involved in tip-stalk cell identity  <cit> .
tip cells express high levels of dll <dig> and vascular endothelial growth factor receptor- <dig> , and have low levels of notch signaling activity  <cit> .
the specification of endothelial cells as tip or stalk cells is transient and its reversibility is contingent on the balance between pro-angiogenic factors.
here we quantify the effect of tgf-β <dig> on in vitro angiogenesis using a matrigel cord formation assay, and determine the impact of this angiogenic cytokine on expression of molecules associated with endothelial tip and stalk cell identity.
methods
cells and culture conditions
primary bovine aortic endothelial cells  were isolated and characterized as described  <cit>  and used between passage  <dig> and  <dig>  cultures were maintained in dulbecco’s modified eagle medium  with 10% fetal bovine serum  and 1 mm sodium pyruvate  at 37 °c in 5% co <dig> and 95% atmospheric air.
confluent monolayers were serum starved 16 h prior to treatment.
tgf-β <dig> dose-response
serum starved baec were treated with  <dig> ,  <dig> ,  <dig> ,  <dig> , or  <dig>  ng/ml of recombinant human  tgf-β <dig>  in serum free dmem for up to 24 h, ± 5 μm sb- <dig> .
cell supernatant  was collected and centrifuged at  <dig> × g for 4 min, and adherent cells lysed for protein or rna analysis.
for some assays, conditioned medium was subjected to additional ultracentrifugation  prior to western blotting.
protein isolation and western blotting
cells were lysed on ice with cell lysis buffer  supplemented with 1% phosphatase inhibitor cocktail  <dig> , 2 μg/ml aprotinin  and 1 mm pmsf .
total protein was quantified using the bio-rad system and electrophoresis was performed with 20–30 μg protein plus β-mercaptoethanol; samples were heated prior to sds-page electrophoresis.
proteins were transferred to pvdf membrane, blocked in 5% skim milk or 5% bsa for 1 h at room temperature, then incubated in primary antibody overnight at 4 °c.
the antibodies used were: mouse monoclonal anti-adam  <dig>  and goat polyclonal anti-adam  <dig> ; both santa cruz biotechnology; rabbit monoclonal anti-endoglin , anti-notch <dig> , anti-vegfr <dig> , anti-pvegfr <dig> tyr  <dig> , rabbit polyclonal anti-hsp <dig>  and anti-psmad <dig> , mouse monoclonal anti-smad <dig> , all from cell signaling; rabbit polyclonal anti-dll <dig>  and anti-mmp- <dig> , rabbit monoclonal anti-vegfr <dig> , all from abcam; anti-α tubulin .
membranes were washed and incubated with a secondary antibody  for 30 min at room temperature.
bands were visualized with luminata classico or luminata forte western hrp substrate  and membranes imaged using a chemidoc mp imaging system .
protein loading was normalized by stripping and re-probing for α-tubulin, followed by densitometry analysis.
rna isolation and gene expression analysis
cells were lysed in ribozol  and rna isolated using aurum total rna columns  according to manufacturers’ protocols.
rna was reverse-transcribed with 4 μl iscript  before amplification using primers for target and housekeeping genes .
ssofast evagreen supermix  was used to determine primer efficiency and for quantitative pcr.
no-template controls and quantitative pcr reactions were run in triplicate with an initial 2 min denaturation at 95 °c, 40 cycles of 95 °c for 5 s, 60 °c for 5 s, followed by melt curve analysis of 65 °c to 95 °c in  <dig>  °c increments on the cfx <dig> realtime system .
full length and soluble splice variant mrna for vegfr <dig> were first normalized to both hprt and gapdh housekeeping genes, and then results from tgf-β1-treated samples were expressed as relative amounts normalized to control  samples.
data were analyzed using cfx manager .table  <dig> sequences of primers used for qpcr
svegfr2a
vegfr2a
hprtb
gapdhb
abovine ortholog of human primer sequences derived from  <cit>
bgenerated using primerblast http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_loc=blasthome
matrigel cord formation assay
the impact of tgf-β <dig> and inhibitors on in vitro angiogenesis was determined by cord formation assay.
10 μl of growth factor containing bd matrigel matrix  was added to each well of cold angiogenesis μ-slides  and allowed to gel at 37 °c for 30 min.
50 μl of baec cell suspension containing  <dig> ×  <dig> cells in media- <dig> made with complete supplements  plus or minus tgf-β <dig> and inhibitors were added to each well.
slides were incubated for 8 h at 37 °c, and one image/well was captured with phase contrast microscopy using a 4x objective.
the generation of cord networks was quantified using the tube formation assay analysis service wimtube ; analyzed parameters were total cord length, # of branching points, and # of loops.
# of tip cells was also determined by manual count of phase contrast images; tip cells were defined as free-ended cells/sprouts with visible filopodia.
statistical analysis
all statistical analysis and graphing were performed using graphpad prism  <dig> software .
means for at least three biological replicates were calculated and plotted with standard error bars.
data were analyzed by using the nonparametric kruskal-wallis test followed by dunn’s post hoc test for multiple comparisons, as appropriate.
differences between means were considered statistically significant when the p value was less than  <dig> .
assays were performed in triplicate unless otherwise indicated.
RESULTS
exogenous tgf-β <dig> induced a dose-dependent decrease in endothelial cord formation on matrigel, with significant reductions in cord length, cord branching, loop formation and tip cell generation detectable with doses of tgf-β1 ≥  <dig>  ng/ml .
western blotting of whole cell lysates from baec treated with exogenous tgf-β <dig> under serum free conditions confirmed our previous findings  <cit>  of significant downregulation of vegfr <dig>  and no alteration in vegfr <dig> expression .
vegfr <dig> was detected as a doublet, with one band approximately 230 kda , and a smaller  putative ‘immature’ isoform  <cit> .
reduction in cell associated vegfr <dig> protein was seen in both mature and immature forms, and was sustained for at least 24 h after removal of exogenous tgf-β <dig> .
there was also significant reduction in the levels of notch <dig> but no changes in its ligand dll <dig> .
tgf-β <dig> induced a profound upregulation of the type iii receptor endoglin  and concomitant activation of smad <dig> signaling, as revealed by enhanced levels of phosphorylated smad <dig>  .
smad <dig> activation was maximal at  <dig>  ng/ml tgf-β <dig>  thus subsequent assays were performed with this concentration.fig.
<dig> tgf-β <dig> induced dose-dependent reduction in endothelial cord formation.
a representative phase contrast images showing endothelial cell cord formation 8 h after plating on matrigel™ in the presence of exogenous tgf-β <dig> .
note higher magnification control panel , showing tip cells  and loop of endothelial cords .
wimtube automated quantification of cord formation showed that tgf-β <dig> significantly inhibits total cord length , cord branching , formation of loops , and generation of tip cells .
tgf-β <dig> doses of  <dig>  ng/ml or higher significantly inhibited cord formation compared to control  or  <dig>  ng/ml.
**p ≤  <dig> ; **p ≤  <dig> ; n = 4; kruskal-wallis test.
scale bars = 300 μm
fig.
<dig> tgf- β <dig> induced dose-dependent changes in endothelial cell receptors.
a representative western blots of endothelial cells exposed to exogenous tgf-β <dig>  for 24 h. densitometry showed significantly reduced vegfr <dig> expression , but no change in vegfr <dig>  in cells treated with higher doses of tgf-β <dig>  the tgf-β <dig> co-receptor endoglin was significantly upregulated in a dose dependent fashion .
notch <dig> was significantly downregulated in endothelial cells treated with  <dig>  and  <dig>  ng/ml tgf-β <dig> , and its ligand dll <dig> showed a trend towards reduced expression with higher doses .
tgf-β <dig> signaling through alk5/smad2-dependent pathways, as revealed by phosphorylation of smad <dig>  was maximal at doses of  <dig>  ng/ml and higher .
*p ≤  <dig> ; **p ≤  <dig> ; n = 3; kruskal-wallis test
the mechanisms of the observed changes in angiogenic signaling pathways were then investigated.
as demonstrated by treatment with the alk <dig> signaling inhibitor sb- <dig> , blockade of alk <dig> signaling significantly eliminated the ability of  <dig>  ng/ml tgf-β <dig> to inhibit matrigel angiogenesis .
sb inhibitor alone had no significant effect compared to dmso vehicle  in these assays.
exogenous tgf-β <dig> led to a reduction in vegfr <dig> phosphorylation  and expression levels, but addition of the sb inhibitor rescued vegfr <dig> signaling .
similar results were obtained using sd- <dig>  another alk <dig> signaling inhibitor .
tgf-β <dig> activation of smad <dig> phosphorylation and endoglin upregulation was also blocked by sb .
notch <dig> levels showed a trend towards significant differences with sb inhibitor , but expression of dll <dig> was not altered .
smad1/ <dig> phosphorylation was also not altered in these samples.
thus, alk <dig> appeared to mediate the effects of tgf-β <dig> on the observed altered angiogenesis signaling pathways.fig.
<dig> tgf-β <dig> induced reduction in endothelial cord formation occurs via an alk <dig> related pathway.
a representative phase contrast images showing endothelial cell cord formation 8 h after plating on matrigel™ under control conditions , 5 μm sb- <dig>  in dmso , 5 ng/ml exogenous tgf-β <dig>  or  <dig>  ng/ml tgf-β <dig> plus 5 μm sb- <dig> .
b wimtube automated quantification of cord formation showed sb inhibitor significantly blocked the ability of  <dig>  ng/ml tgf-β <dig> to significantly inhibit total cord length, cord branching, formation of loops, and generation of tip cells.
sb inhibitor, either alone or in combination with  <dig>  ng/ml tgf-β <dig> was not significantly different from dmso control.
**p ≤  <dig> ; ***p ≤  <dig> ; n = 4; kruskal-wallis test
fig.
<dig> tgf-β <dig> induced reduction in receptor expression occurs via an alk <dig> related pathway.
a) representative western blots and densitometry  from baec exposed to control conditions , 5 μm sb- <dig>  in dmso , 5 ng/ml exogenous tgf-β <dig>  or  <dig>  ng/ml tgf-β <dig> plus 5 μm sb- <dig>  for 24 h. tgf-β <dig> leads reduced vegfr <dig> expression and activation, and significantly upregulated endoglin in an alk5/smad2-dependent fashion .
there were no significant changes in smad1/5-dependent signaling, or in vegfr <dig>  notch <dig> or dll <dig> expression.
*p ≤  <dig> ; n = 3; kruskal-wallis test
the basis of vegfr <dig> downregulation was further investigated.
loss of vegfr <dig> from whole cell lysates of tgf-β <dig> treated baec was accompanied by concomitant increasing levels of full-length protein in serum free conditioned media  collected from the same cells .
qpcr analysis showed that both full-length and the soluble vegfr <dig>  splice variant mrnas were expressed by these cells, with the full-length isoform being the predominant species, expressed approximately  <dig> fold higher than the svegfr <dig> message.
both isoforms of vegfr <dig> message were significantly downregulated in tgf-β <dig> treated cells compared to control.
however, the ratio of full-length to soluble vegfr <dig> mrna was not altered by  <dig>  ng/ml tgf-β <dig> treatment , supporting the finding from fig.
5a that a truncated soluble form of vegfr <dig> protein is not released from these cells upon tgf- β <dig> exposure.fig.
<dig> tgf-β <dig> reduces vegfr <dig> expression via multiple mechanisms.
a western blot showing tgf-β <dig> induced loss of cell associated vegfr <dig> protein and concomitant increase in full-length vegfr <dig> detected in conditioned medium.
b quantitative pcr analysis showed that there was no change in the relative ratio of full length vegfr <dig> mrna to alternative spliced svegfr <dig> mrna upon tgf-β <dig> treatment.
c tgf-β <dig> treatment induced increased expression of adam family sheddase enzyme adam  <dig>  tgf-β <dig> treatment had no effect on the expression of adam  <dig> or the membrane-associated metalloproteinase mmp- <dig>  d ultracentrifugation of endothelial conditioned medium demonstrated that soluble vegfr <dig> is associated with the extracellular vesicle/exosome marker hsp <dig> in tgf-β <dig> treated cells
no change in mmp- <dig>  or adam <dig> protein levels were detected, and adam <dig> was weakly upregulated in tgf-β <dig> treated cells, suggesting enzymatic shedding was not the mechanism for increased cm-associated vegfr <dig> .
in contrast, increased levels of the extracellular vesicle/exosome-associated protein hsp <dig> were also detected in serum-free conditioned medium from tgf-β <dig> treated cells .
these data suggest that in addition to transcriptional downregulation, full-length vegfr <dig> is released from tgf-β <dig> treated endothelial cells as a mechanism to regulate angiogenesis.
discussion
tgf-β <dig> induced a dose dependent inhibition of endothelial cord formation and modulation of endothelial angiogenic receptor expression in an alk <dig> dependent fashion.
we observed enhanced endoglin expression, and reduced notch <dig> and vegfr <dig> expression, which collectively abrogated tip cell formation and stalk elongation.
during sprouting angiogenesis, combined increased vegfr <dig> and reduced notch <dig> signaling lead to gain tip cell phenotype, and increased notch <dig> signaling promotes loss of tip cell phenotype  <cit> .
our results suggest that tgf-β <dig> interferes with tip/stalk cell identity when alk5/smad <dig> signaling pathways are activated.
tgf-β-mediated alk <dig> signaling serves to enhance sprouting angiogenesis in part by indirectly inhibiting tgf-β/alk <dig> signaling  <cit> .
endoglin modulates the balance between alk <dig> and alk <dig> signaling, favouring a pro-angiogenic phenotype  <cit> .
however, in our study, endoglin expression was highest in cells treated with ‘anti-angiogenic’ levels of tgf-β <dig> , which were also associated with alk5/smad <dig> activation.
interestingly, activated  smad1/ <dig> was readily detected independent of tgf-β <dig> treatment.
thus, in our system, tip cell identity may be the default phenotype, which is repressed upon activation of alk5/smad <dig> signaling, possibly via loss of notch  <dig> and vegfr <dig> expression.
we saw concomitant loss of cell-associated full-length vegfr <dig> and increased levels of cm-associated full-length vegfr <dig> upon tgf-β <dig> exposure.
endothelial cells are known to produce an alternatively spliced vegfr <dig> mrna, leading to transcripts lacking the transmembrane domain coded for by exon  <dig>  <cit> .
this transcript codes for a soluble form of the receptor , which could potentially account for the presence of cm-associated vegfr <dig> in our samples.
however, while both full length and alternatively spliced vegfr <dig> transcripts were detectable in baec, the full length vegfr <dig> mrna was by far the predominant isoform, and we found no evidence for a preferential shift in production of message for svegfr <dig> in tgf-β <dig> treated cells.
further, the cm-associated vegfr <dig> was full-length protein as assessed by estimated molecular mass.
thus, the source of vegfr <dig> found in conditioned medium is unlikely to be due to preferential translation of a svegfr <dig> variant mrna upon exposure to tgf-β <dig>
alternatively, vegfr <dig> could be cleaved from the baec cell surface through proteolytic activity.
the matrix metalloproteinase mmp- <dig>  is known to cleave the tgf-β type iii receptor endoglin from endothelial cells, generating a soluble form  <cit> .
in our study, levels of mmp- <dig> were weakly detectable by western blotting, and qpcr revealed cq values >  <dig> .
this, combined with the lack of detectable soluble endoglin in baec conditioned medium, suggests that mmp- <dig> cleavage of surface protein is not a significant determinant of cm-associated vegfr <dig> in our system.
a disintegrin and metalloprotease  family of enzymes act as ‘sheddases’, cleaving surface bound proteins to general soluble isoforms.
adam- <dig> is known to shed vegfr <dig> from endothelial  <cit>  and non-endothelial  <cit>  cell surfaces, and vegfr <dig> is a target of adam- <dig> mediated cleavage in endothelial cells  <cit> .
both adam- <dig> and adam- <dig> were produced by baecs, and there was weak induction of adam- <dig> by tgf-β <dig> in our cells.
however, such sheddase activity would generate cm-associated vegfr <dig> molecules of ~130 kda  <cit> , but we only detected full length  vegfr <dig> in conditioned medium, consistent with an alternative mechanism for release of this receptor from endothelial cells.
recent reports suggest that prolonged surface residence of vegfr <dig> in huvecs leads to protease-mediated cleavage and the generation of a soluble fragment of ~100 kda and a residual cell associated 130 kda fragment  <cit> .
as well, enhanced ubiquitination of vegfr <dig> upon internalization in huvecs leads to endosome-lysosome pathway mediated fragmentation into  <dig> and 120 kda fragments  <cit> .
the absence of such vegfr <dig> fragments in our samples suggests that tgf-β <dig> is not modulating vegfr <dig> levels via these mechanisms in these baecs.
there is growing evidence that vegfr <dig> signals cells in an ‘autocrine’ or ‘intracrine’ fashion, leading to increased receptor activity within the cytosolic  and/or nuclear compartments .
endothelial cells can also release exosomes containing sequestered molecules that then modulate neighbouring cells .
endothelial cells expressing high levels of endoglin also upregulate autophagy  <cit> .
autophagy may enhance extracellular vesicle/exosome release via the mutivesicular body pathway, especially in serum starved endothelial cells  <cit> .
these pathways may also be relevant for tip/stalk cell specification during sprouting angiogenesis, as tips cells display enhanced vegfr <dig> turnover via endocytosis and transfer from early endosomes to multivesicular bodies  <cit> .
further, dll <dig> containing exosomes can be shed from endothelial cells, and dll <dig> containing exosomes modulate tip cell phenotype during sprouting angiogenesis  <cit> .
in agreement with the aforementioned findings, our results extend this activity to include the possibility that shedding of vegfr <dig> containing extracellular vesicles  by vascular endothelium may be enhanced upon exposure to tgf-β <dig>  we detected full length vegfr <dig> in serum free conditioned medium along with the exosome marker hsp <dig>  retinal pigment epithelial cells also shed exosomes containing vegfr <dig> which subsequently modulate endothelial cord formation in vitro  <cit> , but to our knowledge this is the first report that vegfr <dig> might be shed from endothelial cells themselves.
CONCLUSIONS
here we report that tgf-β <dig> alters levels of key angiogenic receptors, and interferes with tip/stalk cell identity when alk5/smad <dig> signaling pathways are activated.
our results suggest that downregulation of surface vegfr <dig> and concomitant increase in vegfr <dig> levels in endothelial cell conditioned medium may be a direct response to alk5-mediated tgf-β signaling.
exosome shedding of vegfr <dig> may rapidly limit the effects of angiogenic stimuli on the cells and stop further sprout formation, but the role of these events in physiological and pathological angiogenesis requires further investigation.
additional files
additional file 1: figure s <dig>  time course of restoration of vegfr <dig> expression.
a) western blot of baec treated with serum free media containing 5 ng/ml tgf-β <dig> for 24 h, followed by recovery in serum free medium lacking tgf-β <dig>  samples were collected after  <dig>   <dig>   <dig> and 72 h recovery.
control culture  was treated with serum free medium lacking tgf-β <dig> for 24 h. b) densitometry of western blots showing significant recovery of vegfr <dig> expression by 48 h post tgf-β <dig> treatment.
* p <  <dig> ; n =  <dig>
additional file 2: figure s <dig>  alk <dig> inhibitor sd- <dig> prevents tgf-β <dig> induced downregulation of vegfr <dig> expression.
western blots showing impact of increases doses of sd- <dig> on baec treated with dmso vehicle  or 5 ng/ml tgf-β <dig> for 24 h.

 the cytokine transforming growth factor beta is a member of the tgf-β superfamily consisting of members including tgf-βs. tgf-β is the predominant and more ubiquitous form. tgf-β ligands signal canonically through type i and type ii serine/threonine kinase receptors, and via the accessory receptor endoglin in vascular endothelial cells. tgf-β binding leads to phosphorylation of intracellular r-smads or, which then complex with co-smad enter the nucleus and associate with transcriptional co-activators or co-repressors to regulate the expression of target genes. tgf-β plays a dual role in angiogenesis by orchestrating a switch from vascular inhibition to pro-angiogenic activity. in particular, there is evidence that tgf-β induced angiogenesis acts with vegf to mediate apoptosis of excessive vascular sprouts and may even be required for initial sprouting from an existing vascular network. the nature of the angiogenic response to tgf-β depends on the balance of alk versus alk signaling input, with alk predominantly promoting sprouting and alk favoring the resolution/stabilization phase of angiogenesis. thus, tgf-β is either pro- or anti-angiogenic, depending on which tgf-rii/smad pathway is engaged. differences also exist in the kinetics and dose responses of these pathways: alk mediated signaling is transient and maximal at low concentrations of ligand while alk mediated signaling is sustained and maximal at higher doses of ligand. increased expression of endoglin led to inhibition of tgf-β/alk signaling in a dose dependent fashion. activation of vascular sprouting during angiogenesis entails the specification of endothelial cells into tip and stalk cells. endothelial tip cells are mainly migratory and polarized with minimal proliferation while stalk cells proliferate throughout sprout establishment and form the nascent vascular lumen cells. the delta-like ligand -notch signaling pathway is involved in tip-stalk cell identity. tip cells express high levels of dll and vascular endothelial growth factor receptor-, and have low levels of notch signaling activity. the specification of endothelial cells as tip or stalk cells is transient and its reversibility is contingent on the balance between pro-angiogenic factors. here we quantify the effect of tgf-β on in vitro angiogenesis using a matrigel cord formation assay, and determine the impact of this angiogenic cytokine on expression of molecules associated with endothelial tip and stalk cell identity. methods cells and culture conditions primary bovine aortic endothelial cells were isolated and characterized as described and used between passage and cultures were maintained in dulbecco’s modified eagle medium with 10% fetal bovine serum and 1 mm sodium pyruvate at 37 °c in 5% co and 95% atmospheric air. confluent monolayers were serum starved 16 h prior to treatment. tgf-β dose-response serum starved baec were treated with,,,, or  ng/ml of recombinant human tgf-β in serum free dmem for up to 24 h, ± 5 μm sb-. cell supernatant was collected and centrifuged at × g for 4 min, and adherent cells lysed for protein or rna analysis. for some assays, conditioned medium was subjected to additional ultracentrifugation prior to western blotting. protein isolation and western blotting cells were lysed on ice with cell lysis buffer supplemented with 1% phosphatase inhibitor cocktail, 2 μg/ml aprotinin and 1 mm pmsf. total protein was quantified using the bio-rad system and electrophoresis was performed with 20–30 μg protein plus β-mercaptoethanol; samples were heated prior to sds-page electrophoresis. proteins were transferred to pvdf membrane, blocked in 5% skim milk or 5% bsa for 1 h at room temperature, then incubated in primary antibody overnight at 4 °c. the antibodies used were: mouse monoclonal anti-adam and goat polyclonal anti-adam; both santa cruz biotechnology; rabbit monoclonal anti-endoglin, anti-notch, anti-vegfr, anti-pvegfr tyr, rabbit polyclonal anti-hsp and anti-psmad, mouse monoclonal anti-smad, all from cell signaling; rabbit polyclonal anti-dll and anti-mmp-, rabbit monoclonal anti-vegfr, all from abcam; anti-α tubulin. membranes were washed and incubated with a secondary antibody for 30 min at room temperature. bands were visualized with luminata classico or luminata forte western hrp substrate and membranes imaged using a chemidoc mp imaging system. protein loading was normalized by stripping and re-probing for α-tubulin, followed by densitometry analysis. rna isolation and gene expression analysis cells were lysed in ribozol and rna isolated using aurum total rna columns according to manufacturers’ protocols. rna was reverse-transcribed with 4 μl iscript before amplification using primers for target and housekeeping genes. ssofast evagreen supermix was used to determine primer efficiency and for quantitative pcr. no-template controls and quantitative pcr reactions were run in triplicate with an initial 2 min denaturation at 95 °c, 40 cycles of 95 °c for 5 s, 60 °c for 5 s, followed by melt curve analysis of 65 °c to 95 °c in  °c increments on the cfx realtime system. full length and soluble splice variant mrna for vegfr were first normalized to both hprt and gapdh housekeeping genes, and then results from tgf-β1-treated samples were expressed as relative amounts normalized to control samples. data were analyzed using cfx manager.table sequences of primers used for qpcr svegfr2a vegfr2a hprtb gapdhb abovine ortholog of human primer sequences derived from bgenerated using primerblast http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_loc=blasthome matrigel cord formation assay the impact of tgf-β and inhibitors on in vitro angiogenesis was determined by cord formation assay. 10 μl of growth factor containing bd matrigel matrix was added to each well of cold angiogenesis μ-slides and allowed to gel at 37 °c for 30 min. 50 μl of baec cell suspension containing × cells in media- made with complete supplements plus or minus tgf-β and inhibitors were added to each well. slides were incubated for 8 h at 37 °c, and one image/well was captured with phase contrast microscopy using a 4x objective. the generation of cord networks was quantified using the tube formation assay analysis service wimtube; analyzed parameters were total cord length, # of branching points, and # of loops. # of tip cells was also determined by manual count of phase contrast images; tip cells were defined as free-ended cells/sprouts with visible filopodia. statistical analysis all statistical analysis and graphing were performed using graphpad prism software. means for at least three biological replicates were calculated and plotted with standard error bars. data were analyzed by using the nonparametric kruskal-wallis test followed by dunn’s post hoc test for multiple comparisons, as appropriate. differences between means were considered statistically significant when the p value was less than. assays were performed in triplicate unless otherwise indicated.  exogenous tgf-β induced a dose-dependent decrease in endothelial cord formation on matrigel, with significant reductions in cord length, cord branching, loop formation and tip cell generation detectable with doses of tgf-β1 ≥   ng/ml. western blotting of whole cell lysates from baec treated with exogenous tgf-β under serum free conditions confirmed our previous findings of significant downregulation of vegfr and no alteration in vegfr expression. vegfr was detected as a doublet, with one band approximately 230 kda, and a smaller putative ‘immature’ isoform. reduction in cell associated vegfr protein was seen in both mature and immature forms, and was sustained for at least 24 h after removal of exogenous tgf-β. there was also significant reduction in the levels of notch but no changes in its ligand dll. tgf-β induced a profound upregulation of the type iii receptor endoglin and concomitant activation of smad signaling, as revealed by enhanced levels of phosphorylated smad. smad activation was maximal at  ng/ml tgf-β thus subsequent assays were performed with this concentration.fig. tgf-β induced dose-dependent reduction in endothelial cord formation. a representative phase contrast images showing endothelial cell cord formation 8 h after plating on matrigel™ in the presence of exogenous tgf-β. note higher magnification control panel, showing tip cells and loop of endothelial cords. wimtube automated quantification of cord formation showed that tgf-β significantly inhibits total cord length, cord branching, formation of loops, and generation of tip cells. tgf-β doses of  ng/ml or higher significantly inhibited cord formation compared to control or  ng/ml. **p ≤ ; **p ≤ ; n = 4; kruskal-wallis test. scale bars = 300 μm fig. tgf- β induced dose-dependent changes in endothelial cell receptors. a representative western blots of endothelial cells exposed to exogenous tgf-β for 24 h. densitometry showed significantly reduced vegfr expression, but no change in vegfr in cells treated with higher doses of tgf-β the tgf-β co-receptor endoglin was significantly upregulated in a dose dependent fashion. notch was significantly downregulated in endothelial cells treated with and  ng/ml tgf-β, and its ligand dll showed a trend towards reduced expression with higher doses. tgf-β signaling through alk5/smad2-dependent pathways, as revealed by phosphorylation of smad was maximal at doses of  ng/ml and higher. *p ≤ ; **p ≤ ; n = 3; kruskal-wallis test the mechanisms of the observed changes in angiogenic signaling pathways were then investigated. as demonstrated by treatment with the alk signaling inhibitor sb-, blockade of alk signaling significantly eliminated the ability of  ng/ml tgf-β to inhibit matrigel angiogenesis. sb inhibitor alone had no significant effect compared to dmso vehicle in these assays. exogenous tgf-β led to a reduction in vegfr phosphorylation and expression levels, but addition of the sb inhibitor rescued vegfr signaling. similar results were obtained using sd- another alk signaling inhibitor. tgf-β activation of smad phosphorylation and endoglin upregulation was also blocked by sb. notch levels showed a trend towards significant differences with sb inhibitor, but expression of dll was not altered. smad1/ phosphorylation was also not altered in these samples. thus, alk appeared to mediate the effects of tgf-β on the observed altered angiogenesis signaling pathways.fig. tgf-β induced reduction in endothelial cord formation occurs via an alk related pathway. a representative phase contrast images showing endothelial cell cord formation 8 h after plating on matrigel™ under control conditions, 5 μm sb- in dmso, 5 ng/ml exogenous tgf-β or  ng/ml tgf-β plus 5 μm sb-. b wimtube automated quantification of cord formation showed sb inhibitor significantly blocked the ability of  ng/ml tgf-β to significantly inhibit total cord length, cord branching, formation of loops, and generation of tip cells. sb inhibitor, either alone or in combination with  ng/ml tgf-β was not significantly different from dmso control. **p ≤ ; ***p ≤ ; n = 4; kruskal-wallis test fig. tgf-β induced reduction in receptor expression occurs via an alk related pathway. a) representative western blots and densitometry from baec exposed to control conditions, 5 μm sb- in dmso, 5 ng/ml exogenous tgf-β or  ng/ml tgf-β plus 5 μm sb- for 24 h. tgf-β leads reduced vegfr expression and activation, and significantly upregulated endoglin in an alk5/smad2-dependent fashion. there were no significant changes in smad1/5-dependent signaling, or in vegfr notch or dll expression. *p ≤ ; n = 3; kruskal-wallis test the basis of vegfr downregulation was further investigated. loss of vegfr from whole cell lysates of tgf-β treated baec was accompanied by concomitant increasing levels of full-length protein in serum free conditioned media collected from the same cells. qpcr analysis showed that both full-length and the soluble vegfr splice variant mrnas were expressed by these cells, with the full-length isoform being the predominant species, expressed approximately fold higher than the svegfr message. both isoforms of vegfr message were significantly downregulated in tgf-β treated cells compared to control. however, the ratio of full-length to soluble vegfr mrna was not altered by  ng/ml tgf-β treatment, supporting the finding from fig. 5a that a truncated soluble form of vegfr protein is not released from these cells upon tgf- β exposure.fig. tgf-β reduces vegfr expression via multiple mechanisms. a western blot showing tgf-β induced loss of cell associated vegfr protein and concomitant increase in full-length vegfr detected in conditioned medium. b quantitative pcr analysis showed that there was no change in the relative ratio of full length vegfr mrna to alternative spliced svegfr mrna upon tgf-β treatment. c tgf-β treatment induced increased expression of adam family sheddase enzyme adam tgf-β treatment had no effect on the expression of adam or the membrane-associated metalloproteinase mmp- d ultracentrifugation of endothelial conditioned medium demonstrated that soluble vegfr is associated with the extracellular vesicle/exosome marker hsp in tgf-β treated cells no change in mmp- or adam protein levels were detected, and adam was weakly upregulated in tgf-β treated cells, suggesting enzymatic shedding was not the mechanism for increased cm-associated vegfr. in contrast, increased levels of the extracellular vesicle/exosome-associated protein hsp were also detected in serum-free conditioned medium from tgf-β treated cells. these data suggest that in addition to transcriptional downregulation, full-length vegfr is released from tgf-β treated endothelial cells as a mechanism to regulate angiogenesis. discussion tgf-β induced a dose dependent inhibition of endothelial cord formation and modulation of endothelial angiogenic receptor expression in an alk dependent fashion. we observed enhanced endoglin expression, and reduced notch and vegfr expression, which collectively abrogated tip cell formation and stalk elongation. during sprouting angiogenesis, combined increased vegfr and reduced notch signaling lead to gain tip cell phenotype, and increased notch signaling promotes loss of tip cell phenotype. our results suggest that tgf-β interferes with tip/stalk cell identity when alk5/smad signaling pathways are activated. tgf-β-mediated alk signaling serves to enhance sprouting angiogenesis in part by indirectly inhibiting tgf-β/alk signaling. endoglin modulates the balance between alk and alk signaling, favouring a pro-angiogenic phenotype. however, in our study, endoglin expression was highest in cells treated with ‘anti-angiogenic’ levels of tgf-β, which were also associated with alk5/smad activation. interestingly, activated smad1/ was readily detected independent of tgf-β treatment. thus, in our system, tip cell identity may be the default phenotype, which is repressed upon activation of alk5/smad signaling, possibly via loss of notch and vegfr expression. we saw concomitant loss of cell-associated full-length vegfr and increased levels of cm-associated full-length vegfr upon tgf-β exposure. endothelial cells are known to produce an alternatively spliced vegfr mrna, leading to transcripts lacking the transmembrane domain coded for by exon. this transcript codes for a soluble form of the receptor, which could potentially account for the presence of cm-associated vegfr in our samples. however, while both full length and alternatively spliced vegfr transcripts were detectable in baec, the full length vegfr mrna was by far the predominant isoform, and we found no evidence for a preferential shift in production of message for svegfr in tgf-β treated cells. further, the cm-associated vegfr was full-length protein as assessed by estimated molecular mass. thus, the source of vegfr found in conditioned medium is unlikely to be due to preferential translation of a svegfr variant mrna upon exposure to tgf-β alternatively, vegfr could be cleaved from the baec cell surface through proteolytic activity. the matrix metalloproteinase mmp- is known to cleave the tgf-β type iii receptor endoglin from endothelial cells, generating a soluble form. in our study, levels of mmp- were weakly detectable by western blotting, and qpcr revealed cq values > . this, combined with the lack of detectable soluble endoglin in baec conditioned medium, suggests that mmp- cleavage of surface protein is not a significant determinant of cm-associated vegfr in our system. a disintegrin and metalloprotease family of enzymes act as ‘sheddases’, cleaving surface bound proteins to general soluble isoforms. adam- is known to shed vegfr from endothelial and non-endothelial cell surfaces, and vegfr is a target of adam- mediated cleavage in endothelial cells. both adam- and adam- were produced by baecs, and there was weak induction of adam- by tgf-β in our cells. however, such sheddase activity would generate cm-associated vegfr molecules of ~130 kda, but we only detected full length vegfr in conditioned medium, consistent with an alternative mechanism for release of this receptor from endothelial cells. recent reports suggest that prolonged surface residence of vegfr in huvecs leads to protease-mediated cleavage and the generation of a soluble fragment of ~100 kda and a residual cell associated 130 kda fragment. as well, enhanced ubiquitination of vegfr upon internalization in huvecs leads to endosome-lysosome pathway mediated fragmentation into and 120 kda fragments. the absence of such vegfr fragments in our samples suggests that tgf-β is not modulating vegfr levels via these mechanisms in these baecs. there is growing evidence that vegfr signals cells in an ‘autocrine’ or ‘intracrine’ fashion, leading to increased receptor activity within the cytosolic and/or nuclear compartments. endothelial cells can also release exosomes containing sequestered molecules that then modulate neighbouring cells. endothelial cells expressing high levels of endoglin also upregulate autophagy. autophagy may enhance extracellular vesicle/exosome release via the mutivesicular body pathway, especially in serum starved endothelial cells. these pathways may also be relevant for tip/stalk cell specification during sprouting angiogenesis, as tips cells display enhanced vegfr turnover via endocytosis and transfer from early endosomes to multivesicular bodies. further, dll containing exosomes can be shed from endothelial cells, and dll containing exosomes modulate tip cell phenotype during sprouting angiogenesis. in agreement with the aforementioned findings, our results extend this activity to include the possibility that shedding of vegfr containing extracellular vesicles by vascular endothelium may be enhanced upon exposure to tgf-β we detected full length vegfr in serum free conditioned medium along with the exosome marker hsp retinal pigment epithelial cells also shed exosomes containing vegfr which subsequently modulate endothelial cord formation in vitro, but to our knowledge this is the first report that vegfr might be shed from endothelial cells themselves.  here we report that tgf-β alters levels of key angiogenic receptors, and interferes with tip/stalk cell identity when alk5/smad signaling pathways are activated. our results suggest that downregulation of surface vegfr and concomitant increase in vegfr levels in endothelial cell conditioned medium may be a direct response to alk5-mediated tgf-β signaling. exosome shedding of vegfr may rapidly limit the effects of angiogenic stimuli on the cells and stop further sprout formation, but the role of these events in physiological and pathological angiogenesis requires further investigation. additional files additional file 1: figure s time course of restoration of vegfr expression. a) western blot of baec treated with serum free media containing 5 ng/ml tgf-β for 24 h, followed by recovery in serum free medium lacking tgf-β samples were collected after and 72 h recovery. control culture was treated with serum free medium lacking tgf-β for 24 h. b) densitometry of western blots showing significant recovery of vegfr expression by 48 h post tgf-β treatment. * p < ; n =  additional file 2: figure s alk inhibitor sd- prevents tgf-β induced downregulation of vegfr expression. western blots showing impact of increases doses of sd- on baec treated with dmso vehicle or 5 ng/ml tgf-β for 24 h. 
the cytokine transforming growth factor beta can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. taken together, our results suggest that endothelial cells exposed to tgf-β lose both tip and stalk cell identity, possibly mediated by loss of vegfr signaling. the role of these events in physiological and pathological angiogenesis requires further investigation. here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to tgf-β under low serum conditions. the process is regulated by soluble factors, principally vascular endothelial growth factor, and via bidirectional signaling through the jagged/notch system, leading to assignment of tip cell and stalk cell identity. this vegfr in conditioned medium was full-length protein and was associated with increased soluble hsp- consistent with a possible shedding of microvesicles/exosomes. tgf-β induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on matrigel, maximal at  ng/ml.
 sprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. the process is regulated by soluble factors, principally vascular endothelial growth factor, and via bidirectional signaling through the jagged/notch system, leading to assignment of tip cell and stalk cell identity. the cytokine transforming growth factor beta can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to tgf-β under low serum conditions.  tgf-β induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on matrigel, maximal at  ng/ml. this occurred via alk5-dependent pathways and was accompanied by significant upregulation of the tgf-β co-receptor endoglin, and smad phosphorylation, but no alteration in smad1/ activation. tgf-β also induced alk5-dependent downregulation of notch but not of its ligand delta-like ligand cell associated vegfr was significantly downregulated and accompanied by reciprocal upregulation of vegfr in conditioned medium. quantitative polymerase chain reaction analysis revealed that this soluble vegfr was not generated by a selective shift in mrna isoform transcription. this vegfr in conditioned medium was full-length protein and was associated with increased soluble hsp- consistent with a possible shedding of microvesicles/exosomes.  taken together, our results suggest that endothelial cells exposed to tgf-β lose both tip and stalk cell identity, possibly mediated by loss of vegfr signaling. the role of these events in physiological and pathological angiogenesis requires further investigation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tip cellvascular sproutingnotch1dll4smad signalingmicroparticlehttp://dx.doi.org/ /501100000015canadian cancer society research institute2012-701069canadian foundation for innovation26472issue-copyright-statement© the author 2017 