BACKGROUND
the role of endothelial progenitor cells  in the formation of new blood vessels has been the focus of intense research and debate over the past years but the involved mechanisms remain unclear.
in pathologic conditions involving tissue ischemia such as myocardial infarction, limb ischemia, and tumor angiogenesis  <cit> , epc are chemotactically recruited from bone marrow and the peripheral blood to the site of injury where they differentiate into mature endothelial cells.
these cells may contribute directly to de novo microvascular blood vessel formation, a process termed vasculogenesis  <cit> .
alternatively, they may act through a more paracrine manner by secretion of proangiogenic growth factors such as vegf rather than integrate directly into newly formed blood vessels,  <cit> .
at the same time, epc recruitment and proangiogenic potential may be adversely influenced by an unfavourable microenvironment  <cit> .
it has been previously shown that chronic inflammation and increased levels of proinflammatory cytokines induce the secretion of the large gtpase guanylate binding protein- <dig>  from endothelial cells.
gbp- <dig> mediates the potent antiangiogenic effects of the cytokines tnf-α, ifn-γ and il- <dig> in vitro and in vivo <cit> .
to investigate the function of gbp- <dig> in epc, we previously established epc stably expressing gbp- <dig>  and performed extensive functional in vitro characterization  <cit> .
premature differentiation as indicated by upregulation of vwf and vegfr- <dig> was observed in these transgenic cells compared to control cells carrying an empty vector only.
furthermore, gbp- <dig> inhibited the proliferation and migration of epc in vitro.
epc were then suspended in a fibrin matrix which was placed in a separation chamber containing a microsurgically created av vessel loop.
the av loop provides a well established and highly standardized in vivo model of predictable blood vessel formation generated from an axial vascular axis  <cit> .
after even distribution of epc in the fibrin matrix, expression of gbp- <dig> prevented cell migration towards the central vessels of the construct compared to control cells  <cit> .
based on these findings, the aim of the current study was to perform further functional testing related to angiogenic capacity of gbp-1-epc in vitro and to investigate the impact of the anti-angiogenic cytokine gbp- <dig> on the morphometry of the tissue-engineered construct as well as its blood vessels in vivo.
RESULTS
vegf secretion in vitro
we quantified the ability of ev epc and gbp- <dig> epc to secrete vegf under incubation with basal medium as well as differentiation medium.
no significant difference was noticed when basal medium was used .
however, differentiation medium induced a significant stimulation of vegf secretion in ev epc and even more so in gbp- <dig> epc .
vegf secretion from gbp- <dig> epc was  <dig> ±  <dig> pg/ml/ <dig> cells, while secretion from ev epc was significantly lower, reaching  <dig> ±  <dig> pg/ml/ <dig> cells .
surgery and animals
all animals tolerated the surgical procedure well without any major complications such as thrombosis of the vascular axis, extrusion of the chambers, infections or hematomas.
patency of the avls was assessed during the explantation after microfil perfusion and during histological examination, revealing no signs of thrombosis.
macroscopic appearance
after explantation, vascularization of matrices was visible by yellow appearing filling of vessels with microfil solution.
macroscopic inspection revealed relatively stable fibrin clots at day  <dig>  there was a slight macroscopic difference between the groups, with the av loops containing ev epc showing slightly more signs of clot degradation compared to epc gbp- <dig> clots.
there was some vascularization of matrices detectable.
all av loops were macroscopically patent upon explantation .
micro-ct
the vascular network could be visualized by micro-ct of the specimens .
in both groups, neovascularisation was evident arising from the main vascular axis.
there was a difference which was particularly evident in the area of the contralateral interpositional vein graft, where new blood vessels appeared more numerous and displayed more advanced growth away from the av loop in the ev epc group .
in contrast, gbp <dig> expression reduced vessel ingrowth away from the av loop .
histology
morphometric analysis was based on histological cross sections of explanted constructs.
we previously demonstrated that vessels perfused with microlfil could be identified histologically by black-appearing filling of the lumina  <cit> .
moreover, the fibrovascular tissue evolving from the two central vessels, artery and vein, could be clearly detected and its margins were easily discernible from the adjacent fibrin matrix.
no infections were noticed macroscopically and histologically as indicated by the absence of inflammatory cells.
apart from the quantitative differences in blood vessel number, construct surface area and blood vessel density there were no differences between the two study groups .
morphometric analysis
there was no significant difference in the numbers of blood vessels per region of interest  between the two groups  .
the total construct surface area measurement of cross sections from ev-epc containing constructs was  <dig>  ±  <dig>  mm <dig>  while there was an average of  <dig>  ±  <dig>  mm <dig> surface area for gbp- <dig> epc.
there was no statistically significant difference  .
the ratio of total blood vessel surface area to total construct surface area as an indicator for blood vessel density within the construct revealed a significant reduction by more than three-fold of the percentage of vascularised area in the gbp-1-epc group compared to control ev epc .
immunohistochemistry and apoptosis
caspase- <dig> staining revealed a significant difference in apoptosis rate, with  <dig> ±  <dig> caspase 3-positive cells/roi in ev epc constructs vs.  <dig> ±  <dig> caspase-3-positive cells/roi in gbp- <dig> epc, indicating a significant reduction in cell apoptosis in the av loop in the presence of gbp- <dig> expression from transplanted epc .
caspase- <dig> positive cells can be identified by their red staining.
fluorescence microscopy
fluorescence microscopy was carried out for constructs containing either gbp- <dig> epc  or ev epc .
two different magnifications show overview  of representative areas for both study groups.
bs- <dig> lectin  was used to label functional blood vessels.
there was an increased number of dii-expressing ev epc  in the close vicinity of small blood vessels  while gbp- <dig> expressing epc were not closely co-localized with blood vessels .
equal distribution was shown for cells containing nuclei by dapi staining .
discussion
the term “therapeutic vasculogenesis” was coined by isner and asahara  <cit>  to imply that de novo blood vessel formation in adulthood can occur from differentiating endothelial progenitor cells derived from the bone marrow and peripheral blood.
in the following years, tremendous efforts have been made in a multitude of experimental and clinical studies using epc to stimulate blood vessel formation in ischemic conditions such as myocardial infarction and limb ischemia  <cit> .
even though a number of clinical trials have demonstrated encouraging results and therapeutic effects of epc in patients with myocardial ischemia  <cit>  and peripheral vascular disease  <cit> , conflicting studies have raised questions regarding mechanisms of action and efficacy of epc in different pathological settings  <cit> .
indeed, it has been shown that epc numbers and function are decreased in conditions such as diabetes  <cit>  and rheumatoid autoimmune disorders  <cit> .
serum levels of proinflammatory cytokines including tnf-α, ifn-γ, and il-1α are increased in patients with rheumatoid arthritis and other autoimmune inflammatory disorders  <cit> .
these cytokines exhibit an inhibitory effect on proliferation and migration of endothelial cells through the gtpase gbp- <dig>  <cit> .
in this context, gbp- <dig> has been identified as a potent anti-angiogenic mediator in colorectal carcinoma, where a positive correlation was found between a high gbp- <dig> expression from tumor endothelial cells and reduced angiogenesis within the tumor  <cit> .
we recently investigated potential effects of gbp- <dig> on endothelial progenitor cell function  <cit>  and aimed to identify antiangiogenic mechanisms that could explain epc dysfunction in the inflammatory state  <cit>  and associated cardiovascular disease  <cit> .
the well characterized murine epc line t17b was stably transfected with gbp- <dig>  compared to t17b expressing empty vector as a control , gbp- <dig> was sufficient to induce premature differentiation in epc, while proliferation and migration were inhibited in vitro <cit> .
in the avl model, gbp- <dig> epc migration towards the central vessels was significantly reduced compared to ev epc controls  <cit> .
in the present study, we further characterized the influence of gbp- <dig> on epc and construct morphometry in a developing three dimensional vascular network in the avl model.
further functional in vitro testing revealed increased vegf secretion from gbp- <dig> epc compared to ev epc under differentiation, while there was no difference in vegf secretion under incubation with basal medium.
hence, vegf secretion is associated with differentiation, which occurs also in vivo when epc differentiate in situ within the construct.
this is a further corroboration of our previous finding that premature differentiation in vivo prevented gbp <dig> epc migration within the avl construct towards the central blood vessels  <cit> .
in vivo, micro ct scans showed neo-vessel formation arising from the main vascular axis in both groups.
this finding was confirmed by morphometric analysis of conventional histological cross sections.
there was visible blood vessel ingrowth towards the center of the construct from the interposed contralateral venous graft in the ev epc group which was not present in the gbp- <dig> epc group.
this may indicate that gbp- <dig> may not only be an intracellular inhibitor of angiogenesis in the endothelial cells itself, but prevent vascular ingrowth towards regions with high levels of gbp- <dig> expression.
even though we noticed increased vegf expression in gbp-1-epc after differentiation in vitro by elisa, the presence of gbp- <dig> in vivo may locally interfere with the proangiogenic stimulus associated with the expression of vegf in our in vivo model.
it may be an unexpected finding that the transplanted gbp-1-epc secrete substantial amounts of vegf in vitro, yet fail to stimulate angiogenesis efficiently in vivo.
we previously demonstrated, however  <cit> , that concomitant gbp- <dig> exression inhibits epc migration towards the site of active angiogenesis in our avl microsurgical model.
this may preclude epc participation in neoangiogenesis since gbp- <dig> are unable to home to the region where they could exhibit an effect on the vascularisation process.
indeed, epc may in this case secrete increased vegf levels, unable to migrate to the site where they could exhibit a biological effect towards angiogenesis.
on the other hand, we demonstrated that epc lacking gbp- <dig> were often located in the close vicinity of newly formed blood vessels, supporting the assumption that they secrete proangiogenic growth factors in vivo, supporting neovascularisation through a paracrine fashion.
our experimental results are supported by clinical findings in colorectal cancer patients where increased gbp- <dig> expression from endothelial cells was associated with a reduction in angiogenesis  <cit> .
we measured a 29% reduction in cross section size of avl constructs containing ev epc compared to gbp- <dig> epc constructs.
our group previously demonstrated reduction of cross section size of the avl constructs when recombinant growth factors vegf and bfgf were suspended within the fibrin matrix, leading to increased angiogenesis and fibrovascular tissue formation  <cit> .
these findings indicate that cross section size of the construct can be used as an indicator for shrinkage due to increased fibrovascular tissue inside formation.
this supports the hypothesis that gbp- <dig> prevented blood vessel formation and hence concomitant degradation of the fibrin construct.
previous work from our group by arkudas et al.
<cit>  demonstrates that fibrin loaded with recombinant vegf does effectively support blood vessel growth in a dose dependent manner.
in this setting, however, vegf is likely homogenously distributed within the fibrin gel, which does not appear top be the case with the transplanted epc in our study.
the presence of gbp- <dig> reduces epc migration capacity, and areas where vegf expression is particulary high do exert a negative chemotactic effect on gbp- <dig> expressing cells, i.e.
gbp-1-epc cannot locate to the vicinity of intense growth of new blood vessels.
to further elucidate the impact of gbp- <dig> on the angiogenic stimulus in our avl model, we assessed the ratio of total blood vessel surface area to total construct surface area in complete representative construct cross sections.
of note, we found a significant reduction in the percentage of blood vessel surface area in relation to the total construct, indicating a reduced vascular density attributable to gbp- <dig> expression from epc.
unfortunately, it is difficult to pinpoint the exact mechanisms by which epc exhibit their effects in this study.
based on the distribution pattern of fluorescently labelled ev-epc, it appears that they exerted their proangiogenic effect mostly in a paracrine fashion rather than being directly integrated into newly formed blood vessels.
overexpression of gbp- <dig> in epc has been shown to reduce their migratory capacity as well as their potential for capillary formation  <cit> .
we also previously confirmed that gbp- <dig> inhibited vessel-directed migration of epc at the tissue level using the rat arterio-venous loop model  <cit> .
hence, we stipulate that several mechanisms contribute to reduced blood vessel formation of gbp- <dig> epc.
on the one hand, it is cell-inherent due to the antiangiogenic effect of gbp- <dig>  on the other hand, gbp- <dig> epc appear to be less capable to localize to areas of intense angiogenesis where they are most needed to exert their function.
it is also known that gbp- <dig> has an anti-apoptotic effect on endothelial cells.
the rate of apoptosis in our construct, as detected by caspase- <dig> staining, was significantly reduced in the gbp- <dig> epc - group.
this may indicate an antiapoptotic effect induced by gbp- <dig>  this hypothesis is supported by findings from pammer et al.
where human umbilical vein endothelial cells  were protected from apoptosis upon transfection with an expression vector for gbp- <dig>  <cit> .
they further attributed the antiangiogenic effect to the fact that gbp- <dig> induces senescence in endothelial cells rather than apoptosis.
CONCLUSIONS
taken together, this is the first study demonstrating the potential of the antiangiogenic proinflammatory large gtpase gbp- <dig> to alter epc behaviour, reduce apoptosis rate and blood vessel density after transplantation into a developing vascular network.
these results may open a new option towards antiangiogenic treatment strategies to inhibit undesired angiogenesis and target inhibition to defined regions of angiogenetic activity in cancer and other disease conditions.

 the role of endothelial progenitor cells in the formation of new blood vessels has been the focus of intense research and debate over the past years but the involved mechanisms remain unclear. in pathologic conditions involving tissue ischemia such as myocardial infarction, limb ischemia, and tumor angiogenesis, epc are chemotactically recruited from bone marrow and the peripheral blood to the site of injury where they differentiate into mature endothelial cells. these cells may contribute directly to de novo microvascular blood vessel formation, a process termed vasculogenesis. alternatively, they may act through a more paracrine manner by secretion of proangiogenic growth factors such as vegf rather than integrate directly into newly formed blood vessels,. at the same time, epc recruitment and proangiogenic potential may be adversely influenced by an unfavourable microenvironment. it has been previously shown that chronic inflammation and increased levels of proinflammatory cytokines induce the secretion of the large gtpase guanylate binding protein- from endothelial cells. gbp- mediates the potent antiangiogenic effects of the cytokines tnf-α, ifn-γ and il- in vitro and in vivo. to investigate the function of gbp- in epc, we previously established epc stably expressing gbp- and performed extensive functional in vitro characterization. premature differentiation as indicated by upregulation of vwf and vegfr- was observed in these transgenic cells compared to control cells carrying an empty vector only. furthermore, gbp- inhibited the proliferation and migration of epc in vitro. epc were then suspended in a fibrin matrix which was placed in a separation chamber containing a microsurgically created av vessel loop. the av loop provides a well established and highly standardized in vivo model of predictable blood vessel formation generated from an axial vascular axis. after even distribution of epc in the fibrin matrix, expression of gbp- prevented cell migration towards the central vessels of the construct compared to control cells. based on these findings, the aim of the current study was to perform further functional testing related to angiogenic capacity of gbp-1-epc in vitro and to investigate the impact of the anti-angiogenic cytokine gbp- on the morphometry of the tissue-engineered construct as well as its blood vessels in vivo.  vegf secretion in vitro we quantified the ability of ev epc and gbp- epc to secrete vegf under incubation with basal medium as well as differentiation medium. no significant difference was noticed when basal medium was used. however, differentiation medium induced a significant stimulation of vegf secretion in ev epc and even more so in gbp- epc. vegf secretion from gbp- epc was ± pg/ml/ cells, while secretion from ev epc was significantly lower, reaching ± pg/ml/ cells. surgery and animals all animals tolerated the surgical procedure well without any major complications such as thrombosis of the vascular axis, extrusion of the chambers, infections or hematomas. patency of the avls was assessed during the explantation after microfil perfusion and during histological examination, revealing no signs of thrombosis. macroscopic appearance after explantation, vascularization of matrices was visible by yellow appearing filling of vessels with microfil solution. macroscopic inspection revealed relatively stable fibrin clots at day there was a slight macroscopic difference between the groups, with the av loops containing ev epc showing slightly more signs of clot degradation compared to epc gbp- clots. there was some vascularization of matrices detectable. all av loops were macroscopically patent upon explantation. micro-ct the vascular network could be visualized by micro-ct of the specimens. in both groups, neovascularisation was evident arising from the main vascular axis. there was a difference which was particularly evident in the area of the contralateral interpositional vein graft, where new blood vessels appeared more numerous and displayed more advanced growth away from the av loop in the ev epc group. in contrast, gbp expression reduced vessel ingrowth away from the av loop. histology morphometric analysis was based on histological cross sections of explanted constructs. we previously demonstrated that vessels perfused with microlfil could be identified histologically by black-appearing filling of the lumina. moreover, the fibrovascular tissue evolving from the two central vessels, artery and vein, could be clearly detected and its margins were easily discernible from the adjacent fibrin matrix. no infections were noticed macroscopically and histologically as indicated by the absence of inflammatory cells. apart from the quantitative differences in blood vessel number, construct surface area and blood vessel density there were no differences between the two study groups. morphometric analysis there was no significant difference in the numbers of blood vessels per region of interest between the two groups. the total construct surface area measurement of cross sections from ev-epc containing constructs was ± mm while there was an average of ± mm surface area for gbp- epc. there was no statistically significant difference. the ratio of total blood vessel surface area to total construct surface area as an indicator for blood vessel density within the construct revealed a significant reduction by more than three-fold of the percentage of vascularised area in the gbp-1-epc group compared to control ev epc. immunohistochemistry and apoptosis caspase- staining revealed a significant difference in apoptosis rate, with ± caspase 3-positive cells/roi in ev epc constructs vs. ± caspase-3-positive cells/roi in gbp- epc, indicating a significant reduction in cell apoptosis in the av loop in the presence of gbp- expression from transplanted epc. caspase- positive cells can be identified by their red staining. fluorescence microscopy fluorescence microscopy was carried out for constructs containing either gbp- epc or ev epc. two different magnifications show overview of representative areas for both study groups. bs- lectin was used to label functional blood vessels. there was an increased number of dii-expressing ev epc in the close vicinity of small blood vessels while gbp- expressing epc were not closely co-localized with blood vessels. equal distribution was shown for cells containing nuclei by dapi staining. discussion the term “therapeutic vasculogenesis” was coined by isner and asahara to imply that de novo blood vessel formation in adulthood can occur from differentiating endothelial progenitor cells derived from the bone marrow and peripheral blood. in the following years, tremendous efforts have been made in a multitude of experimental and clinical studies using epc to stimulate blood vessel formation in ischemic conditions such as myocardial infarction and limb ischemia. even though a number of clinical trials have demonstrated encouraging results and therapeutic effects of epc in patients with myocardial ischemia and peripheral vascular disease, conflicting studies have raised questions regarding mechanisms of action and efficacy of epc in different pathological settings. indeed, it has been shown that epc numbers and function are decreased in conditions such as diabetes and rheumatoid autoimmune disorders. serum levels of proinflammatory cytokines including tnf-α, ifn-γ, and il-1α are increased in patients with rheumatoid arthritis and other autoimmune inflammatory disorders. these cytokines exhibit an inhibitory effect on proliferation and migration of endothelial cells through the gtpase gbp-. in this context, gbp- has been identified as a potent anti-angiogenic mediator in colorectal carcinoma, where a positive correlation was found between a high gbp- expression from tumor endothelial cells and reduced angiogenesis within the tumor. we recently investigated potential effects of gbp- on endothelial progenitor cell function and aimed to identify antiangiogenic mechanisms that could explain epc dysfunction in the inflammatory state and associated cardiovascular disease. the well characterized murine epc line t17b was stably transfected with gbp- compared to t17b expressing empty vector as a control, gbp- was sufficient to induce premature differentiation in epc, while proliferation and migration were inhibited in vitro. in the avl model, gbp- epc migration towards the central vessels was significantly reduced compared to ev epc controls. in the present study, we further characterized the influence of gbp- on epc and construct morphometry in a developing three dimensional vascular network in the avl model. further functional in vitro testing revealed increased vegf secretion from gbp- epc compared to ev epc under differentiation, while there was no difference in vegf secretion under incubation with basal medium. hence, vegf secretion is associated with differentiation, which occurs also in vivo when epc differentiate in situ within the construct. this is a further corroboration of our previous finding that premature differentiation in vivo prevented gbp epc migration within the avl construct towards the central blood vessels. in vivo, micro ct scans showed neo-vessel formation arising from the main vascular axis in both groups. this finding was confirmed by morphometric analysis of conventional histological cross sections. there was visible blood vessel ingrowth towards the center of the construct from the interposed contralateral venous graft in the ev epc group which was not present in the gbp- epc group. this may indicate that gbp- may not only be an intracellular inhibitor of angiogenesis in the endothelial cells itself, but prevent vascular ingrowth towards regions with high levels of gbp- expression. even though we noticed increased vegf expression in gbp-1-epc after differentiation in vitro by elisa, the presence of gbp- in vivo may locally interfere with the proangiogenic stimulus associated with the expression of vegf in our in vivo model. it may be an unexpected finding that the transplanted gbp-1-epc secrete substantial amounts of vegf in vitro, yet fail to stimulate angiogenesis efficiently in vivo. we previously demonstrated, however, that concomitant gbp- exression inhibits epc migration towards the site of active angiogenesis in our avl microsurgical model. this may preclude epc participation in neoangiogenesis since gbp- are unable to home to the region where they could exhibit an effect on the vascularisation process. indeed, epc may in this case secrete increased vegf levels, unable to migrate to the site where they could exhibit a biological effect towards angiogenesis. on the other hand, we demonstrated that epc lacking gbp- were often located in the close vicinity of newly formed blood vessels, supporting the assumption that they secrete proangiogenic growth factors in vivo, supporting neovascularisation through a paracrine fashion. our experimental results are supported by clinical findings in colorectal cancer patients where increased gbp- expression from endothelial cells was associated with a reduction in angiogenesis. we measured a 29% reduction in cross section size of avl constructs containing ev epc compared to gbp- epc constructs. our group previously demonstrated reduction of cross section size of the avl constructs when recombinant growth factors vegf and bfgf were suspended within the fibrin matrix, leading to increased angiogenesis and fibrovascular tissue formation. these findings indicate that cross section size of the construct can be used as an indicator for shrinkage due to increased fibrovascular tissue inside formation. this supports the hypothesis that gbp- prevented blood vessel formation and hence concomitant degradation of the fibrin construct. previous work from our group by arkudas et al. demonstrates that fibrin loaded with recombinant vegf does effectively support blood vessel growth in a dose dependent manner. in this setting, however, vegf is likely homogenously distributed within the fibrin gel, which does not appear top be the case with the transplanted epc in our study. the presence of gbp- reduces epc migration capacity, and areas where vegf expression is particulary high do exert a negative chemotactic effect on gbp- expressing cells, i.e. gbp-1-epc cannot locate to the vicinity of intense growth of new blood vessels. to further elucidate the impact of gbp- on the angiogenic stimulus in our avl model, we assessed the ratio of total blood vessel surface area to total construct surface area in complete representative construct cross sections. of note, we found a significant reduction in the percentage of blood vessel surface area in relation to the total construct, indicating a reduced vascular density attributable to gbp- expression from epc. unfortunately, it is difficult to pinpoint the exact mechanisms by which epc exhibit their effects in this study. based on the distribution pattern of fluorescently labelled ev-epc, it appears that they exerted their proangiogenic effect mostly in a paracrine fashion rather than being directly integrated into newly formed blood vessels. overexpression of gbp- in epc has been shown to reduce their migratory capacity as well as their potential for capillary formation. we also previously confirmed that gbp- inhibited vessel-directed migration of epc at the tissue level using the rat arterio-venous loop model. hence, we stipulate that several mechanisms contribute to reduced blood vessel formation of gbp- epc. on the one hand, it is cell-inherent due to the antiangiogenic effect of gbp- on the other hand, gbp- epc appear to be less capable to localize to areas of intense angiogenesis where they are most needed to exert their function. it is also known that gbp- has an anti-apoptotic effect on endothelial cells. the rate of apoptosis in our construct, as detected by caspase- staining, was significantly reduced in the gbp- epc - group. this may indicate an antiapoptotic effect induced by gbp- this hypothesis is supported by findings from pammer et al. where human umbilical vein endothelial cells were protected from apoptosis upon transfection with an expression vector for gbp-. they further attributed the antiangiogenic effect to the fact that gbp- induces senescence in endothelial cells rather than apoptosis.  taken together, this is the first study demonstrating the potential of the antiangiogenic proinflammatory large gtpase gbp- to alter epc behaviour, reduce apoptosis rate and blood vessel density after transplantation into a developing vascular network. these results may open a new option towards antiangiogenic treatment strategies to inhibit undesired angiogenesis and target inhibition to defined regions of angiogenetic activity in cancer and other disease conditions. 
the goal of the present study was to assess the antiangiogenic capacity of epc expressing gbp- and their impact on blood vessel formation in an axially vascularized 3-d bioartificial construct in vivo. this indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the avl constructs caused by gbp- in addition, gbp- expressed from epc significantly reduced cell apoptosis compared to gbp-1-negative controls. most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of gbp-1-epc. guanylate binding protein- is a large gtpase which is actively secreted by endothelial cells. transgenic epc expressing the proinflammatory antiangiogenic gtpase gbp- can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis. undifferentiated gbp-1-epc, however, did not secrete increased levels of vegf compared to undifferentiated control epc expressing an empty vector. the new vascular network arises from an arterio-venous loop embedded in a fibrin matrix inside a separation chamber. total surface area of the construct as calculated from cross sections was larger after transplantation of gbp-1-epc compared to control ev-epc. this indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing ev-epc. functional in vitro testing demonstrated a significant increase in vegf secretion by gbp-1-epc after induction of cell differentiation. it is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. we previously demonstrated that stable expression of gbp- in murine endothelial progenitor cells induces their premature differentiation and decreases their migration capacity in vitro and in vivo.
 guanylate binding protein- is a large gtpase which is actively secreted by endothelial cells. it is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. we previously demonstrated that stable expression of gbp- in murine endothelial progenitor cells induces their premature differentiation and decreases their migration capacity in vitro and in vivo. the goal of the present study was to assess the antiangiogenic capacity of epc expressing gbp- and their impact on blood vessel formation in an axially vascularized 3-d bioartificial construct in vivo.  functional in vitro testing demonstrated a significant increase in vegf secretion by gbp-1-epc after induction of cell differentiation. undifferentiated gbp-1-epc, however, did not secrete increased levels of vegf compared to undifferentiated control epc expressing an empty vector. in our in vivo experiments, we generated axially vascularized tissue-engineered 3-d constructs. the new vascular network arises from an arterio-venous loop embedded in a fibrin matrix inside a separation chamber. total surface area of the construct as calculated from cross sections was larger after transplantation of gbp-1-epc compared to control ev-epc. this indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing ev-epc. most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of gbp-1-epc. this indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the avl constructs caused by gbp- in addition, gbp- expressed from epc significantly reduced cell apoptosis compared to gbp-1-negative controls.  transgenic epc expressing the proinflammatory antiangiogenic gtpase gbp- can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis. angiogenesisendothelial progenitor cellsguanylate-binding protein 1in vivo tissue engineering 