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An intact VEGF receptor/PI3K/PKB/Akt signaling cascade protects endothelial cells from apoptotic stress-stimuli and mediates the formation of new blood vessels in pathological conditions such as cancer. Therefore, downregulation of this signaling cascade is of clinical interest for antiangiogenic cancer therapy. In this report, we demonstrate that VEGF controls the protein stability of the serine–threonine kinase PKB/Akt via inhibition of PKB/Akt protein degradation. VEGF deprivation or blockage of the VEGF signal transduction cascade with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 resulted in a specific decrease of the PKB/Akt protein level and subsequent cellular restimulation with VEGF rescued its stability.

Real-time quantitative RT–PCR analysis demonstrated that VEGF does not regulate PKB/Akt gene expression. On the other hand, broad range inhibitors of caspases and the proteasome complex prevented VEGF-dependent downregulation of the PKB/Akt protein level indicating that PKB/Akt protein stability is regulated by VEGF-controlled proteolysis. Inhibition of the VEGF receptor and PKB/Akt-downstream PIK-related mTOR-kinase by rapamycin also neutralized the VEGF-protective effect in an PKB/Akt gene expression-independent way but results in proteolysis-dependent reduction of PKB/Akt protein stability. These results demonstrate a novel regulatory mechanism of the activated VEGF receptor/mTOR-signal transduction pathway to control the protein stability of PKB/Akt and survival threshold in endothelial cells. Vascular endothelial growth factor (VEGF) is a major proangiogenic factor in tumor angiogenesis (;; ). It is secreted mainly from tumor and stroma cells and exerts its angiogenic activity through receptors that are almost exclusively located on endothelial cells (EC).

The corresponding VEGF receptor-2 (flk-1/KDR, kinase insert-domain containing receptor) is pivotal for its proangiogenic response because this receptor tyrosine kinase is strongly autophosphorylated on VEGF stimulation and mediates EC mitogenesis and survival (;;,;;; ). The importance of VEGF and KDR for tumor angiogenesis is underlined by the fact that both are expressed in high levels when neovascularization occurs (,; ) and their expression is strongly associated with tumor grade and vascularity in human neoplasms (,;;;; ). Many preclinical studies have demonstrated that VEGF and KDR are promising targets for molecular defined antiangiogenic cancer therapy. Different strategies aim at inhibiting VEGF activity and some of these strategies such as small molecular inhibitors of the VEGF receptor-2 tyrosine kinase or anti-VEGF antibodies are already in clinical trials (;;; ). However, no breakthrough in clinical cancer treatment has been achieved with any antiangiogenic treatment alone so far, but strategies to combine antiangiogenic agents with classic cytotoxic treatment modalities such as chemotherapy or ionizing radiation, seem to be more promising and are now tested both in preclinical and clinical studies (;;;;;;;;; ). On the molecular and cellular level the phosphatidylinositide 3-kinase (PI3K)/Akt signal transduction pathway was identified as an important mediator for proangiogenic signaling and EC survival downstream of KDR.

The serine–threonine kinase Akt, also named PKB, is a proto-oncogenic key player in cellular processes such as cell proliferation, apoptosis, glucose metabolism and cell migration (; ). PKB/Akt is regulated by phosphorylation, which involves the binding of PI3K-phosphorylated phosphoinositides (PI) to the PKB/Akt pleckstrin domain and translocation to the plasma membrane, where PKB/Akt is phosphorylated by the PI-dependent kinase PDK1 and an unidentified kinase referred to as PDK2. Phosphorylation of the two phosphorylation sites Thr308 by PDK1 and Ser473 is required for its activation (,;; ). Further in tumor cells direct degradation of PKB/Akt has also been demonstrated to deregulate PKB/Akt activity and to promote an apoptotic process (,;; ) but such a mechanism has not been identified in endothelial cells so far. VEGF and PI3-kinase-dependent phosphorylation of PKB/Akt suggest an important role of this serine–threonine kinase in endothelial cells downstream of the VEGF receptor. Further transient transfection of HUVE cells with constitutive active PKB/Akt is sufficient to prevent apoptosis in response to serum and VEGF-deprivation while overexpression of a constitutive negative form of PKB/Akt diminishes the VEGF-mediated survival effect. Recently, the importance of PKB/Akt in angiogenesis, EC survival and as a central regulator of an apoptotic threshold was supported in an in vivo zebrafish angiogenesis model.

When treated with the clinically relevant specific VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 zebrafish embryos lacked all major blood vessels at an early time point of embryogenesis. In further developed embryos with already existing major blood vessels PTK787/ZK222584 induced endothelial cell apoptosis.

On the other hand, overexpression of constitutive active PKB/Akt in this in vivo model rescued blood vessel formation in presence of the VEGF receptor tyrosine kinase inhibitor and prevented EC apoptosis. The mammalian target of rapamycin (mTOR) is a downstream entity of the VEGFR/PI3K/PKB/Akt pathway. This serine–threonine-kinase member of the PIK-family recently gained major interest as therapeutic target due to its key regulatory function regulating biogenesis in tumor and untransformed cells by activating p70 S6 kinase (p70 s6k) and inhibiting the translational repressor 4E-BP-1. Interestingly, inhibition of mTOR by rapamycin also potently inhibits angiogenesis and EC proliferation induced by VEGF. The antiangiogenic effect of rapamycin is not only due to decreased VEGF-synthesis but also due to a direct antiproliferative response on VEGF-stimulated ECs (; ). Rapamycin also induces G1 cell cycle arrest or apoptosis in several tumor cell lines dependent on the genetic background of the cell. Rapamycin markedly inhibited tumor growth in preclinical xenograft tumor models and also cooperates with cytotoxic agents as part of combined treatment regimens (;;;; ).

Investigations on EC-survival signaling downstream of the VEGF receptor are of increasing interest in the field of angiogenesis to explore the antiangiogenic mechanism of current strategies on the molecular level. In this report, we investigated the effect of VEGF-induced survival signaling on PKB/Akt protein stability in endothelial cells by two novel antiangiogenic compounds targeting the VEGF receptor/PI3K/PKB/Akt/mTor pathway. VEGF deprivation induces apoptosis and PKB/Akt downregulation in endothelial cells We used a VEGF-dependent in vitro model system of human umbilical vein endothelial (HUVE) cells to investigate the effect of VEGF-signaling on the PI3K/PKB/Akt survival pathway. This system was previously established to characterize VEGF as survival factor.

Illustrates that VEGF (10 ng/ml) prevented cell death of HUVE cells kept under these conditions in which cells were cultured in endothelial cell growth medium and then serum starved in endothelial cell basal medium for 12 and 24 h. Serum starvation induced a substantial amount of apoptosis in these cells, although only 24 h after serum deprivation as determined by trypan blue-exclusion analysis and effector caspase 3-activation. Using Ac-DEVD-pNA as colorimetric caspase 3-substrate, an increase in DEVDase activity was observed 24 h after serum deprivation in absence of VEGF whereas VEGF stimulation prevented effector caspase activation. VEGF deprivation induces cell death and downregulates the Akt protein level in endothelial cells. Serum starvation was performed in endothelial cell basal medium (EBM, 0% serum; 0.1% BSA) in presence or absence of VEGF (10 ng/ml). Cell viability ( a), Caspase 3-like activity ( b) and the PKB/Akt protein level were determined 12 and 24 h after serum deprivation and caspase 3-like activity was measured in cytosolic extracts using Ac-DEVD-pNA as a colorimetric caspase 3 substrate. Absorption values of basal DEVDase-cleavage activity derived from cells grown in normal growth conditions (0 h time point) were taken as baseline.

In ( c), the protein levels of PKB/Akt, PI3K and β-actin were determined 12 and 24 h after change of growth conditions in whole-cell extracts by Western blotting using a polyclonal anti-PKB/Akt, anti-PI3K-p85 and anti- β-actin antibody. For quantification of the PKB/Akt protein level results of three independent immunoblots were analysed with Scion Image computer software. VEGF regulates endothelial cell survival through the activation of the PI3K/PKB/Akt-survival pathway via a multistep post-translational modification process. Inhibition of VEGF-signaling was investigated in this report not on the level of post-translational modification of PKB/Akt but on the level of protein stability of this serine–threonine protein kinase.

Serum deprivation of HUVE cells for 24 h strongly reduced the amount of PKB/Akt protein while neither the total protein level of phosphatidylinositide 3′-kinase (PI3K), which mediates VEGF-induced survival signaling upstream of PKB/Akt, nor the VEGF pathway-independent protein β-actin were affected at this time point. Importantly, VEGF (10 ng/ml) prevented downregulation of the PKB/Akt protein level in these serum-deprived endothelial cells. Immunoblots of total cell lysates were probed with an anti-PKB/Akt antibody that detects the total protein level of all three Akt-isoforms (Akt1-3). Scan analysis of three independent experiments revealed a decrease of the PKB/Akt protein level in absence of VEGF to 50% of the total PKB/Akt protein level detected in presence of VEGF at this time point. Downregulation of PKB/Akt could not be observed at an earlier time point (12 h following start of serum deprivation). The protein level of PKB/Akt was also downregulated at later time points (at 36 and 48 h, respectively) but was difficult to quantify due to the increasing amount of dead cells (data not shown). The survival activity of VEGF is mainly mediated via VEGF receptor-2 (flk-1/KDR) as previously determined with receptor-selective VEGF-mutants that preferentially bind to either one of the VEGF receptors, flk-1/KDR or flt-1 (VEGF receptor-1), respectively.

PTK787/ZK222584 is a clinically relevant antiangiogenic inhibitor of the VEGF receptor tyrosine kinase family and subsequently inhibits VEGF-R-mediated PKB/Akt phosphorylation. On the cellular level, PTK787/ZK222584 decreases endothelial cell proliferation and survival (;; ). To investigate the effect of this antiangiogenic compound on the VEGF-controlled steady-state protein level of PKB/Akt and on cell survival, the protein level of PKB/Akt and the level of cell death was determined in VEGF-supplemented medium in presence of PTK787/ZK222584. Inhibition of KDR activity by PTK787/ZK222584 also resulted in downregulation of the total PKB/Akt protein level and cell death induction to a similar extent as observed on VEGF deprivation.

PTK787/ZK222584-induced caspase-activation was further determined using Ac-DEVD-pNA as colorimetric caspase 3-substrate. Again treatment with PTK787/ZK222584 resulted in a comparable level of caspase-3-like activation as observed on serum deprivation supporting the important role of active VEGF/VEGF-receptor mediated signaling for cell survival. As an independent readout for caspase activation, immunoblotting of cellular extracts was performed using an antibody that only recognizes active caspase-3.

Both serum deprivation and treatment with PTK787/ZK222584 resulted in an increase of the active large p17 fragment and p19 fragment. These immunoblotting experiments indicate that the enzymatic assay for caspase-activity measurement correlates to probing of the active caspase form by Western blotting. Inhibition of VEGF receptor by the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 induces cell death and downregulation of PKB/Akt protein in endothelial cells. HUVE cells were serum-deprived for 24 h in presence or absence of VEGF (10 ng/ml) and PTK787/ZK222584 (100 n M). The PKB/Akt protein level ( a), cell death ( b), caspase 3-like activity ( c) and caspase cleavage ( d) were determined 24 h after serum deprivation. Formation of active caspase 3 was determined by Western blotting using an antibody recognizing the cleaved caspase 3 subunit. Samples were reanalysed with an anti- β-actin antibody to ensure the loading of equal amounts of protein.

VEGF regulates the protein level of PKB/Akt but not the PKB/Akt mRNA level To investigate the regulation of the PKB/Akt protein level by VEGF in more detail, we tested the effect of VEGF-restimulation on PKB/Akt in VEGF-deprived cells. HUVE cells were serum-deprived for 24 h and then restimulated with VEGF for 24 h. Interestingly, the PKB/Akt protein level was again upregulated after treatment with VEGF to a level as observed in cells kept under normal growth medium conditions (EGM). Cells that were VEGF-deprived over the entire 48 h period could not be further analysed for the PKB/Akt protein level due to the massive induction of cell death after this prolonged lack of VEGF and serum (not shown). Addition of VEGF to serum- and VEGF-deprived endothelial cells reupregulates the PKB/Akt protein level independent of the akt transcription (mRNA) level and prevents cells cell death.

HUVE cells were serum-deprived for 24 h and restimulated with VEGF (10 ng/ml) thereafter. PKB/Akt protein ( a) and akt1 mRNA (b) levels were analysed before serum deprivation (growth medium), 24 h after serum deprivation and 24 h after restimulation of serum-deprived HUVEC with VEGF (time point 48 h). In ( a) PKB/Akt protein was detected in whole-cell extracts by Western blotting using an anti-PKB/Akt antibody and samples were reanalysed with an anti- β-actin antibody to ensure the loading of equal amounts of protein. No significant difference of akt1 mRNA-synthesis could be detected with real-time quantitative RT–PCR ( b) between cells growing in growth medium, in serum-deprived cells and in cells that were restimulated with VEGF (10 ng/ml). In ( c) the total amount of living cells was determined 24 and 36 h after serum deprivation in absence and presence of VEGF and in cells that were restimulated with VEGF 24 h after serum deprivation.

Downregulation of PKB/Akt in serum-deprived HUVE cells could be due to a decrease in gene expression (mRNA level) or due to increased protein degradation. Therefore, real-time quantitative RT–PCR of all three ( akt1-3) mRNA isoforms was performed of HUVE cells grown in EC growth conditions (EGM), 24 h after serum deprivation and 24 h after restimulation with VEGF (48 h time point). The calculation of the relative RNA amounts was based on the CT-values of akt1-3 mRNA (standard curve method) and ribosomal RNA was used as internal standard. In HUVE cells, akt1 mRNA was 4 times more prevalent than akt 2 or 3 mRNA in all tested samples (data not shown). Any extensive change of the PKB/Akt protein level would therefore most probably be due to transcriptional changes of akt1. However, no significant differences could be detected between the akt1 mRNA or akt2 and akt3 mRNA (data not shown) of cells grown in serum-rich conditions (EGM) and the respective mRNA levels of cells cultured under serum-free conditions in absence or presence of VEGF. Thus, abrogation of VEGF-signaling through growth factor deprivation and/or VEGF receptor tyrosine kinase inhibition controls the PKB/Akt protein level but not via VEGF-mediated PKB/Akt gene expression.

Restimulation of serum-deprived cells resulted in an upregulated PKB/Akt-protein level. We also determined the effect of VEGF-addition on the cellular recovery following 24 h serum deprivation. Prolonged serum deprivation for an additional 8 h resulted in a drastic reduction of living cells as determined by trypan blue exclusion. On the other hand, VEGF-stimulation of serum-starved cells prevented this loss of living cells and stabilized the cellular population at a level observed with cells that were continuously cultured in presence of VEGF. Again the number of cells that were VEGF-deprived for over 32 h was difficult to quantify due to rapid increase of dead cells and cellular debris after prolonged VEGF-deprivation. Caspase- and proteasome-dependent decrease of PKB/Akt protein level To test for VEGF-dependent PKB/Akt protein degradation, HUVE cells were preincubated with the broad range caspase inhibitor ZVAD-fmk (20 μ M) or the proteasome inhibitor MG-262 (4 n M) prior to VEGF-deprivation.

Both ZVAD-fmk and MG-262 prevented downregulation of the total PKB/Akt protein level , indicating that caspase activity and the proteasome participate in the process leading to PKB/Akt degradation in response to abrogation of VEGF-signaling. In absence of these inhibitors caspase 3-like activity was strongly induced after VEGF-deprivation at the time point of extended PKB/Akt degradation. On the other hand, DEVDase activity was low in cells pretreated with VEGF or the broad range caspase inhibitor ZVAD-fmk. Control experiments demonstrated that ZVAD-fmk and MG-262 alone do not affect the PKB/Akt protein level under optimal growth conditions and when tested in absence of stress (data not shown). Interestingly, cells pretreated with the proteasome inhibitor MG-262 displayed only low caspase 3-like activity indicating that a proteasome-regulated step is induced most probably prior to DEVDase activation.

To determine whether Akt-degradation could be a direct consequence of enhanced caspase activity the PKB/Akt protein level was probed following addition of recombinant active caspase-3 to the cellular extracts. Demonstrates that the PKB/Akt level was reduced in presence of recombinant active caspase-3, which was prevented by coadministration of the specific caspase-3 inhibitor Z-DVED-fmk (100 μ M).

Quantification of treatment-dependent cell death revealed that both the broad range caspase inhibitor and the proteasome inhibitor prevented to a large extent VEGF-deprivation-induced cell death. Thus, these results demonstrate that VEGF-mediated signaling regulates the protein level of PKB/Akt by specific control of PKB/Akt protein degradation and suggest that VEGF-dependent PKB/Akt-stabilization represents an additional control level for this cell survival pathway. PKB/Akt protein is degraded in a caspase- and proteasome-dependent way. HUVE cells were cultured for 24 h in presence or absence of VEGF (10 ng/ml), the broad range caspase inhibitor ZVAD-fmk (20 μ M) or the proteasome inhibitor MG262 (4 n M). The PKB/Akt protein level was detected in whole-cell extracts by Western blotting using an anti-PKB/Akt antibody ( a) and samples were reanalysed with an antibody against β-actin to ensure the loading of equal amounts of protein. Caspase 3-like activity was determined in cytosolic extracts using Ac-DEVD-pNA as a colorimetric caspase 3-substrate ( b).

In ( c) cytosolic extract of HUVEC grown in EGM conditions was incubated with recombinant caspase 3 (5 U/ μl) in presence or absence of the caspase 3 inhibitor Z-DEVD-fmk (100 μmol). The PKB/Akt protein level was analysed 1.5 h after addition of recombinant caspase 3 by Western blotting and compared to the β-actin protein level.

In ( d), cell viability was determined by trypan blue exclusion. Dominant active PKB/Akt prevents VEGF-dependent cell death Serum deprivation and inhibition of the VEGF-receptor by PTK787/ZK222584 downregulates the active state of the PI3K/Akt-survival pathway and results in PKB/Akt protein degradation. To further determine the role of this pathway for serum-deprivation-induced cell death, we infected endothelial cells with high-titer retroviruses encoding an empty control construct or HA-tagged wildtype PKB/Akt or constitutively dominant active myrPKB/Akt. At 24 h following infection, HUVECs or retroviral-infected HUVECs were serum deprived for 24 h and the amount of dead cells were determined by trypan blue exclusion. Similar to VEGF-treatment expression of dominant-active PKB/Akt prevented serum deprivation-dependent cell death.

Enforced expression of wildtype PKB/Akt partially reduced the amount of cell death in the serum-starved endothelial cell population. Immunoblotting of cell extracts from several independent experiments revealed that the wildtype PKB/Akt-construct was always expressed to a higher extent in infected cells in comparison to the myrPKB/Akt-construct. Nevertheless, phosphorylation of Akt Ser-473 was only detectable in cells expressing this constitutively active PKB/Akt construct and only cells expressing dominant-active PKB/Akt were fully stress-resistant.

These results suggest that prolonged suspension of the VEGF-mediated survival signal might activate an autoregulatory proapoptotic feedback mechanism and that both active PKB/Akt and the protein level of PKB/Akt regulate this apoptotic threshold. Inhibition of mTOR by rapamycin induces PKB/Akt degradation in presence of VEGF The serine–threonine kinase mTOR has recently gained major interest as therapeutic target due to its regulatory function downstream of VEGF-controlled PKB/Akt. Inhibition of mTOR with the immunosuppressive macrolide rapamycin has a drastic antiproliferative effect in both tumor and endothelial cells and rapamycin is currently investigated for its antiangiogenic properties alone and in combined treatment modalities (;;; ). Currently, two rapamycin analogues, CCI-779 and RAD001, are undergoing clinical evaluation in different human cancers trials. We tested whether inhibition of this downstream element of the VEGFR-PI3K/Akt pathway also modulates PKB/Akt protein stability considering that mTOR is a major regulator of protein synthesis in mammalian cells.

First, we analysed the inhibitory effect of rapamycin and PTK787/ZK222584 on mTOR in presence of VEGF with a site-specific antiphospho-p70 s6k antibody to detect the Thr389 phosphorylation status of p70 s6k, an endogenous substrate of mTOR. VEGF strongly enhanced the p70 s6k Thr389 phosphorylation level (lane 2) but p70 s6k phosphorylation was completely abrogated when cells were pretreated with rapamycin (lane 4). Of note, cellular pretreatment with PTK787/ZK222584 to a large extent also suppressed p70 s6k phosphorylation indicating that inhibition of the VEGF-receptor still controls this further downstream signaling element (lane 3). HUVE cells were preincubated with rapamycin (1 μg/ml) prior to VEGF stimulation of serum-deprived cells and total PKB/Akt protein levels were analysed by immunoblotting 24 h thereafter. Treatment with rapamycin resulted in a specific decrease of the PKB/Akt protein level in these VEGF-stimulated endothelial cells similar if not even to a more extended degree as to the response of complete VEGF-deprivation or abrogation of VEGF-signaling by PTK787/ZK222584 (, compare with and ).

Reduction of the protein level was specific for PKB/Akt and did not represent an overall cellular response since both PI3K- and β-actin-protein levels were not affected by rapamycin treatment. Similar to the experiments on the level of the VEGF receptor real-time quantitative RT–PCR of akt1 mRNA of cells treated with rapamycin in presence of VEGF revealed that decrease of PKB/Akt protein level could not be linked to reduced gene transcription. On the other hand, pretreatment of HUVE cells with ZVAD-fmk or MG-262 abrogated treatment-dependent decrease of the total PKB/Akt protein level. Comparing the similar effect of VEGF receptor- and mTOR-inhibition these results suggest that mTOR is the responsible downstream mediator for VEGF-controlled PKB/Akt protein stability. Inhibition of mTOR by rapamycin inhibits VEGF-signaling downstream of PKB/Akt and induces caspase- and proteasome-dependent reduction of PKB/Akt protein level. ( a) Following 12 h serum deprivation, HUVE cells were stimulated with VEGF (10 ng/ml) and after 30 min, the activity status of p70 S6 kinase was analysed in whole-cell extracts from cell treated with either PTK787/ZK222584 (100 nm) or rapamycin (1 μ M) by Western blotting with an antiphospho-p70S6K (Thr389) antibody.

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(The phosphorylated band marked with an asterisk represents an unspecific crossreaction with the antibody.) ( b) The PKB/Akt protein level was determined in whole-cell extracts of HUVE cells grown for 24 h in strictly VEGF-dependent conditions extracts in absence or presence of rapamycin, using an anti-PKB/Akt antibody. Samples were reanalysed with an anti- β-actin antibody. ( c) Quantitative real-time RT–PCR ( akt1 mRNA) of HUVE cells grown for 24 h in strictly VEGF-dependent conditions in presence or absence of rapamycin (1 μg/ml). (d) HUVE cells were grown for 24 h in strictly VEGF-dependent conditions in presence or absence of rapamycin, the caspase inhibitor ZVAD-fmk (20 μ M) or the proteasome inhibitor MG262 (4 n M). The PKB/Akt protein level was analysed using an anti-PKB/Akt antibody. All experiments were carried out independently at least three times. Caspase- or proteasome-mediated degradation of PKB/Akt by rapamycin Rapamycin has a strong inhibitory effect on VEGF-mediated proliferation (; ).

To determine rapamycin-induced caspase induction, DEVDase activity was determined in cellular extracts derived from VEGF-stimulated cells treated with rapamycin (1 μg/ml). In comparison to DEVDase activation in cells that were treated with the VEGF receptor inhibitor PTK787/ZK222584, induction of caspase 3-like activity was lower in response to rapamycin.

Analysis of caspase 3 by immunoblotting with antiactive caspase 3 antibodies revealed that rapamycin also induces processing of the caspase zymogen to its active form. Nevertheless, a smaller amount of dead cells was determined in response to rapamycin treatment than in response to VEGF-deprivation at this time point. Caspase-dependent degradation of PKB/Akt by rapamycin might represent only a secondary mechanism for the high degree of rapamycin-mediated PKB/Akt-degradation (see ) and rapamycin-induced cell death or apoptosis could be delayed. Direct targeting of PKB/Akt to the proteasome could also be an important mechanism for rapamycin-induced Akt-degradation, as also suggested by the effect of MG-262 on rapamycin-induced Akt-degradation. To detect ubiquitinated forms of PKB/Akt, we performed immunoprecipitation experiments with antibodies against PKB/Akt with subsequent detection of the ubiquitinated protein with an antiubiquitin-specific antibody.

Immunoprecipitation was performed 24 h following treatment with rapamycin or PTK787/ZK222584 in presence of the proteasome inhibitor MG-262 (4 n M, added 4 h prior to cell lysis to prevent rapid degradation of ubiquinated protein by the proteasome). Western-blot analysis of immunoprecipitates from HUVE cell extracts revealed a drastic increase of ubiquitinated proteins (Mw60 kDa) detected in anti-PKB/Akt immunoprecipitates derived from rapamycin- and PTK787/ZK222584-treated cells, but only low amounts of ubiquitinated proteins were detected in immunoprecipitates from control cells. Although we cannot exclude that other proteins than PKB/Akt were ubiquitinated and coimmunoprecipitated with the anti-Akt antibody. Inhibition of mTor by rapamycin induces endothelial cell death caspase 3 activation and treatment-dependent protein ubiquitination.

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After serum-deprivation, HUVEC were kept in strictly VEGF-dependent conditions in presence of either PTK787/ZK222584 (100 n M) or rapamycin (1 μ M). Caspase 3-like activity ( a), cell viability ( b) and caspase cleavage ( c) were determined at the 24 h time point. Caspase 3-like activity was measured in cytosolic extracts using Ac-DEVD-pNA as a colorimetric caspase 3 substrate, formation of active caspase 3 was analysed by Western blotting using an antibody against cleaved caspase 3.

( d) Immunoprecipitation of PKB/Akt was performed in total cell lysates derived from control (lane 1), PTK787/ZK222584- (lane 2) and rapamycin (lane 3)-treated cells using anti-PKB/Akt antibody-linked agarose beads at 4°C overnight. Immunoprecipitates were subjected to Western blotting and the same samples were probed with antibodies against PKB/Akt and ubiquitin. The relevance of the PKB/Akt protein kinase for cellular survival has been investigated in different cell types and animal systems and in response to many stress factors. In this report, we demonstrate that VEGF not only mediates its survival signal via this serine–threonine kinase but also protects the integrity of this important signaling entity. Eventually, antiangiogenic compounds targeting VEGF-signaling exert their effect via disruption of this pathway on these two levels.

VEGF-deprivation or blockage of the VEGF-signal transduction cascade resulted in specific decrease of the PKB/Akt protein level and subsequent cellular restimulation with VEGF rescued its stability. Real-time quantitative RT–PCR determined that the level of PKB/Akt gene expression is not diminished by lack of VEGF. On the other hand, broad range inhibitors of caspases and the proteasome complex prevented a reduction of the PKB/Akt protein level in response to VEGF-deprivation indicating that VEGF protects PKB/Akt from stress-induced degradation. Interestingly, inhibition of the PIK-related mTOR-kinase which is located downstream of the VEGF-regulated PI3K/Akt-pathway, also neutralized this VEGF-protective effect. Thus, VEGF protects PKB/Akt from degradation by keeping the PI3K/Akt/mTor pathway in its active state while inhibition of this cell survival stimulus and subsequent Akt degradation might promote a proapoptotic feedback loop (see below). We observed induction of DEVDase activity and loss of cell viability 24 h following VEGF-deprivation in analogy to previous studies demonstrating VEGF and the VEGF-activated PI3K/Akt survival pathway to protect endothelial cells from stress-induced apoptosis (; ).

Interestingly, VEGF controlled the integrity of PKB/Akt not via the rate of PKB/Akt gene expression to sustain intact survival signaling but prevented its degradation. Further restimulation of serum-deprived cells with VEGF again upregulated the PKB/Akt protein level independent of its rate of synthesis and rescued the stress-exposed cellular population. Inhibition of the proteasome or the caspase machinery prevented PKB/Akt protein degradation and suppressed cell death indicating that both proteolytic machineries are involved in this stress response. The proteasome inhibitor also abrogated DEVDase activation demonstrating that the proteasome-induced step occurs prior to caspase activation. Although we also detected direct PKB/Akt ubiquitination or at least interaction with proteasome-targeted proteins in response to VEGF-deprivation which indicates an immediate involvement of the proteasome in PKB/Akt protein degradation. PKB/Akt represents an important target for controlled proteolysis in the cell killing process of tumor cells and different modes of stress-dependent PKB/Akt degradation were previously observed. Proteasome-dependent PKB/Akt degradation is induced in tumor cells treated with the Hsp90-specific inhibitors geldanamycin, 17-AAG and radicicol and concomitantly sensitizes these cells to proapoptotic stimuli (; ).

On the other hand, caspase-dependent PKB/Akt cleavage is induced in response to integrin clustering in p53-wildtype tumor cells, and caspase cleavage-resistant PKB/Akt mutants protected cells from apoptosis in response to matrix detachment (, ). Indeed, PKB/Akt is most probably also a direct, specific substrate for caspases in endothelial cells as observed in this report in presence of recombinant caspase 3. Furthermore, reactive oxygen species-induced PKB/Akt degradation was suggested to occur via a multistep caspase-dependent and caspase-independent process involving multiple proteases.

In this report, we delineate that VEGF-deprivation and cellular treatment with specific inhibitors of VEGF-signaling, such as PTK787/ZK222584 and rapamycin, result in specific loss of PKB/Akt in endothelial cells and indicate the relevance of caspase and proteasome-dependent PKB/Akt degradation. Expression of dominant-active PKB/Akt prevented stress-induced cell death which was only partially the case for enforced expression of wildtype PKB/Akt. An activated VEGF-receptor-PKB/Akt-axis could be part of an important surveillance mechanism in endothelial cells and at the same time relevant for a high apoptotic threshold preventing stress-induced cell death. However, prolonged suspension of the VEGF-mediated survival signal will activate a proapoptotic feedback loop and will eventually induce complete switching off through degradation of PKB/Akt.

Such a possible scenario might include primary proteasome-dependent processing of inactive PKB/Akt and activation of the apoptotic caspase machinery followed by caspase-dependent PKB/Akt degradation. Here, initial PKB/Akt degradation could partially decrease an apoptotic threshold to further promote full activation of the apoptotic machinery. Such a sequential process could explain that inhibition of proteasome activity prevented caspase activation and that both proteasome and caspase inhibitors rescued loss of PKB/Akt protein.

In comparison to cells of lymphoid origin or with an apoptosis-prone tumor cell background serum-starvation-induced apoptosis in endothelial cells is delayed. During the first 24 h of serum deprivation the amount of living cells was only reduced to a level of 80% in comparison to cells that were continuously stimulated with VEGF. A drastic reduction of living cells only occurred thereafter which could still be prevented by restimulation with VEGF at the 24 h time point. At this time point though we already observed a specific reduction of the PKB/Akt-protein-level. The cellular rescue can neither be reconciled with a cell population that already has a fully activated apoptotic program nor with a rapid VEGF-driven proliferation of a putative remaining cell population over this short time period. Inactivation followed by degradation of PKB/Akt are both part of a slowly progressing loss of the cellular apoptotic threshold guardian PKB/Akt eventually leading to endothelial cell death upon continued stress.

Interestingly, treatment of endothelial cells with the macrolide rapamycin induced a similar effect on PKB/Akt stability as VEGF deprivation and inhibition of the VEGF receptor tyrosine kinase by PTK787/ZK222584. However, inhibition of the downstream kinase mTOR resulted only in partial caspase activation, even at high concentration of rapamycin. Consequently, a smaller amount of dead cells was observed in response to rapamycin treatment in comparison to VEGF-deprivation or in response to treatment with PTK787/ZK222548.

This might be due to a delayed or diminished apoptotic response upon treatment with rapamycin in addition to a treatment induced-G1 arrest (;;;; ). VEGF-deprivation and/or inhibition of the upstream VEGF receptor interrupts multiple cell survival and growth promoting signaling pathways emanating from the VEGF-receptor and further receptor tyrosine kinases. This will induce an enhanced or accelerated stress response leading to cell death, in comparison to the signal inhibition by rapamycin which is limited to processes downstream of mTOR. Nevertheless, the PKB/Akt protein level was strongly decreased in response to treatment with rapamycin and thus potent degradation of PKB/Akt by inhibition of mTOR strongly suggests that mTOR is a downstream mediator of VEGF-controlled PKB/Akt protein stability. Preclinical studies revealed that inhibition of VEGF-dependent signal transduction cascades with pharmacological compounds is a potent antiangiogenic strategy alone and in particular when combined with a cytotoxic treatment modality. On the molecular level the PI3K/PKB/Akt pathway is activated by growth factors and is important for cellular survival in response to multiple stress factors (;;;;; ).

Not surprisingly, this pathway is mutated in different tumor entities leading to a growth advantage during carcinogenesis and to treatment resistance due to a high PI3K/PKB/Akt-mediated survival threshold. In endothelial cells, the integrity of the PI3K/PKB/Akt pathway is protected by VEGF through an enhanced stability of Akt, as demonstrated in this report. Thus, endothelial cells of the angiogenic tumor system are still responsive to pharmacological compounds on a post-translational level leading to reduced enzyme activity and in turn resulting in reduced protein stability. Hence, reduction of PKB/Akt protein stability in endothelial cells represents an additional level for the cooperative effect of a combined treatment modality enhancing the antiangiogenic and overall treatment response.

Cell cultures Primary cultures of human umbilical vein endothelial cells (HUVEC, Promo Cell, Heidelberg, Germany) were used between the fourth and the seventh passage and cultured at 37°C and 5% CO 2 atmosphere in endothelial cell growth medium kit (EGM, PromoCell, Heidelberg) containing 5% fetal calf serum, 0.4% ECGS/H, 0.1 ng/ml EGF, 1 ng/ml bFGF, 1 μg/ml hydrocortisone, 50 ng/ml amphotericin B and 50 μg/ml gentamicin. For all experiments, except retroviral infection, HUVEC were plated in 10 cm Petri dishes at a density of 1.5 × 10 5 cells/dish and were allowed to attach for 24 h. Before use, the tissue culture dishes were coated with 1.5% gelatin and washed with PBS buffer.

For growth factor starvation, cells were washed once with PBS and then incubated with endothelial cell basal medium (EBM, PromoCell Heidelberg) supplemented with 0.1% bovine serum albumin, amphotericin B and penicillin/streptomycin. Human VEGF 165 (R&D Systems) was added to EBM to a final concentration of 10 ng/ml.

Preincubation with PTK787/ZK222584 (100 n M, Novartis Pharma Inc.) was performed for 2 h and preincubation with ZVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone, an irreversible inhibitor of caspases 1, 3, 4 and 7, 20 μ M, Biomol)), the proteasome inhibitor MG-262 (Z-Leu-Leu-Leu-B(OH) 2, 4 n M, Biomol) and rapamycin (1 μg/ml, Sigma) was performed for 1 h prior to the respective medium change and continued thereafter. Western blotting and immunoprecipitation HUVEC were washed once with PBS and then detached from the Petri dish using trypsin/EDTA.

For Western blotting whole-cell lysis was performed in 1% Triton/PBS supplemented with protease inhibitors (5 μg/ml pepstatin A, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml DTT and 1 m M PMSF) for 15 min on ice. Cell lysates were immediately frozen and stored at −80°C.

The protein concentration was determined with the BioRad DC-Protein assay. Cellular proteins (30–50 μg) were resolved by SDS–PAGE and blotted onto PVDF membranes. Membranes were probed with anti-PKB/Akt, antiphospho- PKB/Akt (Ser-473), anti-p70s6K, antiphospho- p70s6K (Thr389), anticleaved caspase-3 (Cell Signaling), anti-PI3K-p85 (Upstate Biotechnology), anti-HA-tag (Roche Biochemicals) and anti- β-actin-antibodies (Sigma). Antibody detection was achieved by ECL-enhanced chemiluminescence (Amersham) according to the manufacturer's protocol. Quantification of the blots was performed with Scion Image computer software.

All experiments were carried out independently at least three times. For immunoprecipitation of ubiquitinated Akt, the proteasome inhibitor MG-262 (4 n M) was added 4 h prior to cell harvesting.

Cell lysis was performed in 1% NP40, 5% Glycerol, 1 M Tris-HCl, 5 M NaCl, 0.5 M KCl, 100 m M MgCl 2, 100 m M CaCl 2, 5 m M EDTA supplemented with protease inhibitors and the proteasome inhibitor MG-262 (4 n M) at 4°C for 15–30 min. After homogenization by douncing 15 times with a 25G needle, cell lysates were centrifuged at 7000 g for 10 min. Protein concentrations were adapted (BioRad DC-Protein assay) and 400–500 μg of protein was incubated overnight with 6 μg/sample slurry anti-Akt Ph domain-linked agarose-beads (Upstate Biotechnology) at 4°C.

In parallel, aliquots of cell lysates were stored for Western blotting as a control of PKB/Akt downregulation, total protein amounts and detection of ubiquitinated proteins in total cell lysates. Immune complexes were washed four times with the lysis buffer, and immunoprecipitates and total cell lysates were subjected to Western blotting and probed with antibodies against PKB/Akt (Cell Signaling) and ubiquitin (Santa Cruz).

Detection of akt1-3 mRNA by real-time quantitative RT–PCR Total RNA was isolated from cells by use of the Rneasy Mini Kit (Quiagen, Basel, Switzerland). For cDNA synthesis of extracted RNA (0.5 μg RNA per sample to be analysed), Taqman Reverse Transcription Reagents (including random hexamers) were used as described in the users manual of the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). This system was used for real-time monitoring of PCR amplification of the cDNA following the Taqman Universal PCR Master Mix Protocol (Applied Biosystems) (; ).

The amplification of Akt1-3 cDNA was performed using the following forward (fw) and reverse (rev) primer and Taqman probes (tp): Akt1-fw: 5′-CCACCTGACCAAGATGACAGC, Akt1-rev: 5′-TGGCCGAGTAGGAGAACTGG, Akt1-tp: 5′-AGCGAGCGCAGGCCCCACTT, Akt2-Fw: 5′-CCAAG GAGGTCATGGAGCA, Akt2-Rev: AGGAGCTTCTTCTG GACCACG, Akt2-tp: 5′-AGGTTCTTCCTCAGCATCAAC TGGCAGG, Akt3-Fw: 5′-GGCACTCCAGAATATCTGGCA, Akt3-Rev: 5′-CCCTAG GCCCCACCAGTCTA. Akt3-tp: 5′-CAGAGGTGTTAGAAGATAATGACTATGGCCGAG C. These experiments were performed twice and repeated with commercially available akt1 primers and probe (Applied Biosystems). All probes were labeled at the 5′end with the reporter dye molecule 6-carboxy fluorescein (FAM) and at the 3′-end with the quencher dye molecule 6-carboxy-tetramethyl-rhodamine (TAMRA). As internal standard ribosomal RNA (Applied Biosystems) was amplified separately as indicated in the Ribosomal RNA Control Reagents protocol. The standard curve method was used for relative quantification of akt1 mRNA. In all experiments, input control (rRNA) showed no significant difference in input.

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. Part of the book series (NSSA, volume 208) Abstract The blood vessels in and around tumors can be distinguished from normal vasculature in several ways. Tumor-associated vessels are derived from surrounding host blood vessels as a result of neovascularization ( Folkman, 1985) and are typically more permeable than normal vessels ( Dvorak et al., 1988, Gerlowski and Jain,1983). These observations lead to the hypothesis that tumors can alter blood vessel function by producing regulator substances.

A polypeptide regulator known as vascular permeability factor (VPF) or vascular endothelial growth factor (VEGF) has now been purified and found to display a surprisingly wide spectrum of vascular activities, some of which are pro-inflammatory in nature. In addition to synthesis by tumor cells, VPF has now been found to be a product of monocytes and lymphocytes. Taken together, these results suggest that VPF may be a new inflammatory cytokine.