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(Circulation. 1996;94:1647-1654.)
© 1996 American Heart Association, Inc.

Articles

Functional Upregulation of the Vascular Endothelial Growth Factor Receptor KDR by Hypoxia

Johannes Waltenberger, MD; Ulrike Mayr; Siegwald Pentz, PhD; Vinzenz Hombach, MD

the Department of Internal Medicine II (Cardiology) (J.W., U.M., V.H.) and the Department of Medical Genetics (S.P.), Ulm (Germany) University Medical Center.

Correspondence to Johannes Waltenberger, MD, Department of Internal Medicine II (Cardiology), Ulm University Medical Center, Robert-Koch-Str 8, D-89081 Ulm, Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Vascular endothelial growth factor (VEGF) is a specific endothelial mitogen and chemoattractant that has been shown to be useful for inducing therapeutic angiogenesis in ischemic myocardium and found to stimulate mitogenicity and chemotaxis of endothelial cells through the receptor tyrosine kinase KDR. Although VEGF expression is upregulated by hypoxic stimuli, regulation of KDR remained unknown under these conditions.

Methods and Results With the use of human umbilical vein endothelial cells and transfected porcine aortic endothelial cells, KDR protein was found to be upregulated under hypoxic conditions (2% O₂) in both cell types. This process of KDR upregulation was found to be reversible, was maximal after 24 hours of hypoxia, and was regulated on a posttranscriptional level. Furthermore, the susceptibility for VEGF-induced mitogenicity was enhanced under hypoxic conditions as shown by [³H]-thymidine incorporation assay. The activated state of increased VEGF function in hypoxic endothelial cells was associated with elevated tyrosine phosphorylation of KDR as demonstrated by anti-phosphotyrosine blot.

Conclusions These data indicate that hypoxia stimulates VEGF-dependent signaling not only by upregulation of VEGF ligand but also by functional upregulation of a specific signaling receptor. Therefore, these data provide evidence that the endothelium plays an active role in hypoxia-induced angiogenesis.

Key Words: endothelium • growth substances • hypoxia • receptors • signal transduction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Hypoxia is a state of reduced oxygen supply to tissue; if accompanied by inadequate removal of metabolites secondary to reduced tissue perfusion in contexts such as arteriosclerosis or arterial embolism, the condition is defined as ischemia. Ischemia of solid organs such as heart or skeletal muscle is a well-known stimulus for the formation of collateral circulation, resulting in compensatory adaptation of tissue perfusion. Molecular concepts for these processes are beginning to emerge.^{1 2} Hypoxia and ischemia represent adequate stimuli for the upregulation of angiogenic factors such as VEGF.^{3 4 5 6 7} With different animal models, the therapeutic application of VEGF to ischemic myocardium, called "therapeutic angiogenesis," results in the stimulation of collateral formation and enhancement of collateral blood flow.⁸ Functional benefits of VEGF-induced myocardial angiogenesis could be quantified recently in terms of improvement of myocardial function (ejection fraction) and reduction of left ventricular infarct size.⁹ Therefore, VEGF-induced myocardial angiogenesis may be of benefit in humans, in whom other means of revascularization are not applicable. In fact, based on in vivo data obtained from an animal model of limb ischemia, the first human gene therapy trial in the cardiovascular system is currently under way, aiming at an increased VEGF expression in ischemic areas of occluding peripheral artery disease.¹⁰

VEGF,^{11 12} also known as vascular permeability factor,^{13 14} is an endothelium-specific mitogen. Besides stimulation of angiogenesis in vivo,^{15 16} VEGF has been shown to stimulate endothelial proliferation in vitro¹⁷ and in vivo^{18 19} as well as endothelial chemotaxis,²⁰ vascular permeability,¹⁷ endothelium-derived relaxing factor–dependent vasodilatation,²¹ and thrombogenicity.²² Recently, two receptors for VEGF have been identified. Both KDR²³ and Flt-1,²⁴ two receptor tyrosine kinases, bind VEGF with high affinity. Although KDR can transduce signals for mitogenicity, chemotaxis, and cytoskeletal reorganization,²⁰ the function of Flt-1 remains unclear. The mechanism of signal transduction through both receptors remains largely unknown.²⁰ Most recent data from gene knockout experiments suggest that both Flt-1 and Flk-1, the mouse homologue of KDR, are crucial components of the embryonic development of the vascular system, with Flt-1 regulating the interaction of endothelial cells with their environment²⁵ and Flk-1 believed to play a central role in endothelial cell differentiation.²⁶ In the adult organism, it is anticipated that KDR might play an important role in the regulation of endothelial function and angiogenesis, and its alteration could result in endothelial dysfunction.²⁰

Thus far, little is known about the functional regulation of KDR and Flt-1 under pathological conditions both in vivo and in vitro. The mRNAs encoding KDR and Flt-1 have been shown to be detectable in malignant gliomas^{27 28} and tissues directly adjacent to the tumor margin,²⁹ whereas no signal could be detected in normal brain tissue. Furthermore, both messages could be detected in other tumors, namely capillary hemangiomas³⁰ and liver metastases from human colon cancer.³¹ However, nothing is known about the regulation of VEGF receptor protein and possible mechanisms of regulation.

The aim of the present study was to analyze the expression and function of KDR under hypoxic conditions. We have tried to determine whether reduced oxygen tension affects VEGF-dependent endothelial regulation and function. The results obtained significantly broaden our knowledge of the molecular basis of angiogenesis, which has important implications for both the concepts of tumor angiogenesis and therapeutic angiogenesis. Moreover, these data raise the possibility that the VEGF/KDR system may function as a survival mechanism for salvaging transiently ischemic tissue.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cells, Cell Culture, and Reagents
HUVE and PAE cells and PAE cells overexpressing KDR (PAE/KDR) or Flt-1 (PAE/Flt-1) were used as described before.²⁰ PAE cells were cultured in Ham's F-12 medium (Biochrom) supplemented with 10% FCS and penicillin-streptomycin. HUVE cells were cultured in MCDB107 or EBM (PromoCell) supplemented with endothelial cell supplement ECGS/Heparin (PromoCell) and 10% FCS. Cell culture was carried out in humidified incubators (Heraeus) at 37°C in the presence of 5% CO₂ and an O₂ content of either 21% (room air) or 2% (lowered by displacement with N₂). The cell density in the culture dishes was assessed quantitatively by use of an automated cell counter (CASY I, Scharfe System). Recombinant VEGF₁₆₅ was a kind gift of D. Gospodarowicz (Chiron Corp). A polyclonal rabbit antiserum against KDR (NEF) was used as described before.²⁰ Recombinant PDGF-BB protein was expressed in the yeast strain Saccharomyces cerevisiae,³² and the R₃ antiserum³³ was used to immunoprecipitate the PDGF ß-receptor in PAE/PDGF-ßR cells as described before.³⁴ For detection and immunoprecipitation of MAP kinase, the monoclonal antibody anti-PAN ERK (Affiniti) was applied. The monoclonal horseradish peroxidase–conjugated phosphotyrosine antibody RC20H (Affiniti) was used to detect phosphotyrosine. For demonstration of ongoing translation processes in cultured cells, protein synthesis was inhibited with cycloheximide (Sigma Chemicals) at a concentration of 10 µg/mL. For detection of the KDR message in Northern blot hybridization, a 3-kb Sma I fragment of KDR cDNA²³ was used, and the Flt-1 mRNA was specifically detected with a 4.5-kb EcoRI/Nsi I fragment of the Flt-1 cDNA.²⁴ For estimation of the RNA amount, a human GAPDH probe³⁵ was applied. An RNA ladder marker (GIBCO BRL) was used to estimate transcript sizes.

Mitogenicity Assay
HUVE and PAE/KDR cells were seeded sparsely in 12-well culture dishes (Nunc). The complete medium (see above) was changed to serum-free medium supplemented with 0.01 mg/mL BSA on the following day and changed again after an additional 24 hours of incubation. Cells were stimulated with VEGF for 20 hours in the presence of 0.25 µCi [³H]-thymidine/mL (Amersham) and 1 µmol/L unlabeled thymidine. High-molecular-weight ³H-radioactivity was precipitated by use of 5% ice-cold trichloroacetic acid and was quantified by liquid scintillation counting.

Assessment of Protein Expression
Subconfluent HUVE or PAE/KDR cells (25 cm²) were cultured under different conditions and lysed in a Triton X-100 buffer. KDR receptors were immunoprecipitated with the NEF antiserum, and MAP kinase was immunoprecipitated with the use of the anti-PAN ERK monoclonal antibody. Immunoprecipitates were washed three times with decreasing salt concentrations, run out on a 7.5% SDS-PAGE, and blotted onto a nitrocellulose membrane (Hybond C extra, Amersham). Detection of proteins was performed by hybridization with the specific antiserum and visualization with a chemiluminescence-based detection system (ECL, Amersham) and autoradiography (Hyperfilm MP, Amersham).

Receptor Autophosphorylation in Intact Cells
Subconfluent HUVE or PAE/KDR cells (75 cm²) were incubated for 20 hours in medium containing 1% FCS. After 5 minutes of preincubation with 100 µmol/L Na₃VO₄, PAE/KDR cells were stimulated with 50 ng/mL VEGF for 8 minutes at 37°C, and HUVE cells were stimulated with 3 ng/mL of VEGF under the same conditions. Cells were solubilized in NP-40 (1%) lysis buffer. Lysates were used for immunoprecipitation with the antisera (NEF or anti-PAN ERK monoclonal antibody for PAE/KDR and HUVE cells, respectively), and the samples were subjected to SDS-PAGE (7.5% for receptor analysis and 12% for MAP kinase analysis) and blotted onto a nitrocellulose membrane (Hybond C extra, Amersham). Phosphorylated proteins were detected by immunoblotting with the horseradish peroxidase–conjugated phosphotyrosine antibody RC20H (Affiniti), followed by the application of a chemiluminescence-based detection system (ECL, Amersham) and autoradiography. In the case of PAE/PDGF-ßR cells for analysis of the PDGF ß-receptor, PDGF-BB (50 µg/mL) was used for stimulation of cells and the R₃ antiserum was used to immunoprecipitate the receptor.

In Vitro Immune Complex Kinase Assay
Cell culture and immunoprecipitation were performed essentially as previously described.²⁰ After 5 minutes of preincubation with 100 µmol/L Na₃VO₄, cells were stimulated with 50 ng/mL VEGF for 8 minutes at 37°C and solubilized in a lysis buffer containing 1% CHAPS. The cell lysates were subjected to immunoprecipitation with the antiserum NEF, followed by an immune complex kinase reaction (7 minutes at 20°C in the presence of [-³²P]ATP). The samples were separated by SDS-PAGE (7.5%), and the proteins were cross-linked to the gel by a 30-minute incubation in 2.5% glutaraldehyde, followed by fixation in 10% acetic acid–40% methanol. Finally, gels were further treated for 1 hour at 55°C in 1 mol/L KOH to remove serine-bound phosphate.³⁶ Dried gels were exposed to Hyperfilm MP (Amersham). Bands corresponding to KDR were quantified by use of phosphostorage technology (Fuji).

RNA Extraction and Northern Blotting
mRNA was extracted from cultured cells using the guanidinium isothiocyanate–based MicroFastTrack kit (InVitrogen). Denatured mRNA (2 µg) was run out on a 1.0% agarose gel containing 6.6% formaldehyde and subsequently transferred by capillary electrophoresis in 20x SSC buffer (1x SSC is 0.15 mol/L NaCl and 0.015 mol/L Na₃ citrate, pH 7) to a nylon membrane (Hybond N, Amersham). The filter was probed with [³²P]-labeled (Megaprime DNA labeling system, Amersham) cDNAs. Hybridization was carried out at 42°C for 16 hours in 5x SSPE, 50% formamide, 0.1% SDS, and 5x Denhardt's solution³⁷ with 10⁶ cpm/mL labeled probe. Final washing of the filter was carried out at 52°C in 0.1x SSC, 0.1% SDS. Autoradiography was performed at -80°C with Hyperfilm MP (Amersham). Furthermore, signals were quantified with phosphostorage technology (Fuji).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Hypoxia Stimulates KDR Expression on a Posttranscriptional Level
HUVE cells and PAE/KDR cells express functional KDR receptors. The mature receptors migrate in SDS-PAGE corresponding to a molecular size of 205 kD, whereas the precursors migrate slightly <200 kD (Fig 1). When oxygen tension is reduced from room air (21% O₂) down to hypoxic conditions (2% O₂), the expression of KDR protein is significantly upregulated in both cell lines tested (Fig 1). The upregulation of KDR protein can be clearly detected after 12 hours of hypoxia, reaches a maximum at 24 hours of hypoxia, starts to decline at 36 hours, and could not be detected any more after 48 hours of hypoxia (data not shown). Inhibition with cycloheximide (10 µg/mL) during the hypoxic period leads to complete loss of KDR protein, indicating de novo synthesis of KDR during the hypoxic period of 24 hours (Fig 1). When the hypoxic stimulus is followed by a 6-hour period of reoxygenation, ie, incubation of the cells at a normal oxygen tension (21% O₂), the upregulation of mature receptor protein is partially reversible in both cell types. In PAE/KDR cells, a smaller fragment of KDR can be detected next to the precursor and the mature protein (Fig 1). This fragment, however, does not become phosphorylated, and its nature has not been further characterized.

View larger version (66K): [in this window] [in a new window]   Figure 1. Expression of KDR receptor protein in PAE/KDR and HUVE cells. Influence of hypoxia, reoxygenation, and inhibition of protein synthesis. Confluent monolayers of both cell types were cultured under normal (21% O2) or hypoxic (2% O2) conditions for defined time periods and lysed. KDR-specific immunoprecipitation with the NEF antiserum was followed by SDS-PAGE (7.5%) and Western blot analysis by use of a nitrocellulose membrane, the primary antiserum NEF, and a chemiluminescence-based detection system for visualization. Protein synthesis was inhibited with cycloheximide (10 µg/mL). Similar results were obtained in at least five independent experiments.

VEGF stimulation of PAE/KDR cells or HUVE cells results in the phosphorylation of the mature KDR protein, which can be demonstrated both in the intact cell by Western blot analysis by use of a phosphotyrosine-specific antiserum (Fig 2) and by an in vitro kinase assay (Fig 3). In PAE/KDR cells stimulated with 50 ng/mL VEGF, tyrosine phosphorylation of KDR is enhanced under hypoxic conditions. This increase in the phosphorylation state was significant in the intact cell (Fig 2). When quantitatively assessed by in vitro kinase assay, the level of tyrosine phosphorylation of VEGF-stimulated KDR was fourfold in the hypoxia-exposed PAE/KDR cells compared with cells under normoxic conditions (Fig 3). The enhancement of tyrosine phosphorylation under hypoxic conditions was found to be partially reversible after 6 hours of reoxygenation, ie, 56% of the level of the hypoxic cells (Fig 3). In HUVE cells, a stimulatory effect of hypoxia on VEGF-induced KDR autophosphorylation also could be found. This was true, however, at lower concentrations of VEGF such as 3 ng/mL (Figs 2 and 3). Hypoxia itself did not induce KDR phosphorylation in the absence of VEGF (data not shown). When the PDGF ß-receptor is expressed and studied in the same cellular background (PAE/PDGF-ßR), no such hypoxia-related increase in PDGF-ßR protein (data not shown) or increase in PDGF-BB–induced tyrosine phosphorylation could be observed (Fig 4). The results displayed in Figs 1 through 4 were highly reproducible, and similar results could be found in at least three independent experiments.

View larger version (53K): [in this window] [in a new window]   Figure 2. Influence of hypoxia on tyrosine phosphorylation of KDR in intact PAE/KDR and HUVE cells. Confluent monolayers were stimulated with VEGF for 8 minutes at 37°C (50 ng/mL for PAE/KDR cells; 3 ng/mL for HUVE cells). Lysates were immunoprecipitated with the KDR-specific antiserum NEF, separated by SDS-PAGE (7.5%), and blotted onto a nitrocellulose membrane. Phosphotyrosine-containing proteins were detected by hybridization with an anti-phosphotyrosine antibody (RC20H) and a chemiluminescence-based detection system. Similar results were obtained in three independent experiments.

View larger version (66K): [in this window] [in a new window]   Figure 3. In vitro phosphorylation of KDR in PAE/KDR and HUVE cells. Cell monolayers were stimulated with VEGF (50 ng/mL for PAE/KDR cells; 3 ng/mL for HUVE cells) and lysed. KDR-specific immunoprecipitation with the NEF antiserum was followed by an in vitro kinase assay; samples were analyzed by SDS-PAGE (7.5%) followed by KOH treatment of the gel and autoradiography on Hyperfilm MP. Virtually identical results were obtained in three unrelated experiments.

View larger version (42K): [in this window] [in a new window]   Figure 4. Influence of hypoxia on tyrosine phosphorylation of the PDGF ß-receptor in intact PAE/PDGF-ß-R cells. The experiment was performed as described for Fig 2. PDGF-BB was used to stimulate cells; the antiserum R3 was used to immunoprecipitate the PDGF ß-receptor.

When both PAE/KDR and HUVE cells were used, Northern blot analysis for KDR did not reveal any increase in mRNA levels induced by either 4 or 24 hours of hypoxia (Fig 5, left). In fact, there appears to be a decrease in mRNA encoding KDR. In PAE/KDR cells, a 4.5-kb species could be detected, corresponding to the transcribed fragment of KDR cDNA, that had been transfected into these cells. In HUVE cells, a 7.0-kb transcript was found. In addition to KDR, HUVE cells also express the second VEGF receptor Flt-1 (Fig 5, right). Two major transcripts of 8.0 and 7.5 kb could be detected as a doublet. Analogous to the data obtained for KDR, no significant change in the Flt-1 mRNA level could be found after 24 hours of hypoxia.

View larger version (113K): [in this window] [in a new window]   Figure 5. Northern blot analysis of KDR and Flt-1 in PAE/KDR and HUVE cells. Left, Denatured mRNA (2 µg) was run out on a 1% agarose gel containing 6.6% formaldehyde and subsequently blotted onto a nitrocellulose membrane. Before RNA extraction, cells were cultivated under normal oxygen content (21% O2; tracks 1 and 4) or hypoxic conditions (2% O2; tracks 2, 3, 5, and 6) for up to 24 hours. 32P-labeled human KDR cDNA was used for hybridization. The autoradiograph was exposed for 7 days at -80°C, and transcript sizes are indicated on the left (left-hand panel). The same filter is shown below, this time hybridized with a 32P-labeled human GAPDH cDNA probe for estimation of RNA loading. Right, Similar experiment as before; however, hybridization in the upper panel was performed with a 32P-labeled human Flt-1 cDNA probe.

During our observation periods of up to 48 hours, the number of endothelial cells per culture dish remained unaffected by hypoxia (data not shown). Therefore, changes in receptor expression represent true changes in the number of receptors per cell.

VEGF Acts as a Survival Factor for Hypoxic Endothelium
Hypoxia negatively affected baseline mitogenicity of endothelial cells. A 24-hour period of hypoxia reduced basal DNA synthesis to about 50% to 80% of the value obtained under normal oxygen tension (21% O₂), which was true for both HUVE and PAE/KDR cells (data not shown). However, VEGF at concentrations <1.0 ng/mL could significantly stimulate DNA synthesis in both cell types, and this stimulation was more efficient under hypoxic conditions compared with the stimulation under normoxic (21% O₂) conditions (Fig 6). For PAE/KDR cells, this effect could also be seen at VEGF concentrations >1 ng/mL. Therefore, VEGF can revert the negative effect of hypoxia on mitogenicity of the endothelium. In contrast, PDGF ß-receptor expressing PAE cells failed to respond to stimulation with PDGF-BB under hypoxic conditions (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 6. Mitogenicity assay. PAE/KDR (A) and HUVE (B) cells were grown under normoxic conditions in 12-well tissue culture plates to subconfluence in the presence of 10% FCS and serum starved for 40 hours. Cells were stimulated with VEGF for 24 hours under either normoxic () or hypoxic () conditions. Incorporation of [3H]-thymidine is shown vs the unstimulated control. Data represent the mean±SEM of three independent experiments, each performed in triplicate.

MAP Kinase Is a Component of the KDR-Dependent Signal Transduction Cascade But Is Not Involved in the Hypoxia-Inducible Stimulation of Mitogenicity
MAP kinase was found to be involved in the signal transduction cascade secondary to VEGF stimulation of KDR. VEGF stimulates tyrosine phosphorylation of MAP kinase (p42^mapk), whereas the protein level of MAP kinase remained unaffected. The phosphorylation of MAP kinase is not further enhanced after 24 hours of cultivation of PAE/KDR cells under hypoxic conditions (Fig 7). On the other hand, VEGF stimulation of Flt-1–expressing PAE/Flt-1 cells did not result in stimulation of MAP kinase phosphorylation (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 7. Assay of MAP kinase phosphorylation. A subconfluent monolayer of transfected PAE cells expressing KDR was stimulated with 50 ng/mL VEGF for 8 minutes at 37°C, lysed, and subjected to MAP kinase–specific immunoprecipitation. Samples were analyzed by Western blot with use of the phosphotyrosine-specific antibody RC20H (A) and an antiserum directed against MAP kinase for specificity control and for quantification of protein content (B). Specifically bound antibodies were detected by chemiluminescence.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
To the best of our knowledge, this is the first report on the functional regulation of mature KDR protein, a signal transducing receptor for VEGF. VEGF ligand is known to be upregulated under hypoxia in vitro, correlating to the stimulation of angiogenesis in vivo.^{3 27} Here, we have presented data on the functional upregulation of KDR under conditions of hypoxia in vitro. Endothelial cells, ie, the target of VEGF, show an improved response under hypoxic conditions, establishing a novel and additive mechanism of an important biological principle with direct therapeutic implications.

The expression of KDR protein was found to be upregulated under hypoxic culture conditions in two different types of endothelial cells, ie, HUVE and PAE cells carrying a construct, which leads to KDR expression under the control of the human cytomegalovirus promoter (PAE/KDR cells). The expression of KDR protein was found to be transiently upregulated by hypoxia in both cell types, whereas the relative amount of specific KDR mRNA levels remained stable or even decreased in either situation, indicating a posttranscriptional upregulation of the receptor level independent of the KDR promoter.

This putative regulatory mechanism is different from the hypoxia-induced upregulation of the ligand VEGF in stromal cells,^{3 4} which is the result of both transcriptional activation and increased mRNA stability.⁷ The mechanism of action of KDR upregulation has not been further elucidated. It is conceivable, however, that the efficiency of translation or posttranslational modifications could be favored under reduced oxygen tension, which may have to do with changes in the formation of free radicals. In fact, the hypoxia-induced posttranscriptional upregulation of protein levels in endothelial cells has been reported before. Cyclooxygenase-1 protein and function were found to be elevated in pulmonary endothelial cells as early as 15 minutes after reduction of oxygen tension, pointing toward a posttranscriptional mechanism.³⁸ Thus far, the majority of data available on the regulation of protein expression under hypoxic conditions have been obtained on soluble molecules; membrane-bound receptor tyrosine kinases have not yet been studied in detail. Therefore, it is an interesting possibility that internalization of membrane-bound proteins such as KDR may be reduced under hypoxic conditions, resulting in enhanced protein stability.

Under hypoxic conditions, a significantly stronger VEGF-induced mitogenic response can be observed in both HUVE and PAE/KDR cells compared with normoxic controls. It also is noteworthy that the basal DNA turnover, ie, the [³H]-thymidine incorporation rate, is somewhat lower under hypoxia. However, VEGF can fully compensate the lowered basal DNA turnover. In some instances, VEGF appears to stimulate DNA synthesis in hypoxic cells even above the level of normoxic cells. Therefore, VEGF may function as a survival factor for transiently hypoxic endothelium. In fact, recent data establish VEGF as a survival factor for endothelial cells in vivo.³⁹ Taken together, elevated levels of KDR in hypoxic endothelial cells may help to ensure an adequate endothelial response to VEGF.

In PAE/KDR cells, tyrosine phosphorylation of KDR- and VEGF-induced mitogenicity is strongly enhanced under hypoxic conditions. Increased tyrosine phosphorylation of KDR can be explained, at least in part, by the elevated level of receptor protein. It is an interesting possibility, however, that the phosphorylation of the individual KDR molecule also is enhanced. Similar effects of an enhanced VEGF-inducible signal could also be demonstrated in HUVE cells, although HUVE cells showed a weaker response to hypoxia, and this response could be observed at 10-fold lower VEGF concentrations compared with PAE/KDR cells. This is in accordance with previously published data.²⁰ In this context, it is noteworthy that HUVE cells express significant levels of Flt-1, a high-affinity VEGF receptor with a comparably weak tyrosine kinase activity and an as-yet-unidentified function in the differentiated organism, whereas PAE/KDR cells lack this receptor.²⁰ Therefore, the shift of the dose-response curve to lower VEGF concentrations in HUVE cells could be a result of the formation of Flt-1/KDR heterodimers with a predicted higher affinity compared with KDR homodimers. However, the functional existence of Flt-1/KDR heterodimers remains to be demonstrated. On the other hand, the weaker response of VEGF stimulation under hypoxic conditions in HUVE cells could also be explained by a potential scavenger function of Flt-1.

No change in Flt-1 mRNA levels under hypoxic conditions could be found in the two cell lines studied. At the present time, it is technically not possible to investigate whether the Flt-1 protein level changes under hypoxic conditions because the sensitivity of reagents currently available does not allow detection of small differences.

Very recently, increased binding of VEGF to cultured bovine retinal endothelial cells under hypoxic conditions was described.⁴⁰ In light of our data, the observation by Thieme et al⁴⁰ might reflect an increased expression of KDR protein. Tuder et al⁴¹ described an upregulation of KDR mRNA in a model of chronic lung hypoxia in vivo. In that case, paracrine stimulation of the endothelium may contribute to changes in KDR mRNA in vivo, which could not be observed in the single-cell culture used in our study. After submission of our manuscript, additional data concerning a paracrine upregulation of KDR became available.⁴² In our article, however, we have shown the functional regulation of VEGF receptor activity under hypoxic conditions, including the mechanism leading to an increased mitogenic response of the endothelium.

Until recently, no information about signal transduction of KDR was available. The first piece of evidence was the involvement of GAP and members of the Src family of cytoplasmic tyrosine kinases.²⁰ In this article, we were able to demonstrate tyrosine phosphorylation of MAP kinase (p42^mapk), which is severalfold induced on VEGF stimulation of KDR on intact cells. This strongly suggests that p42^mapk is involved in the signal transduction cascade of the VEGF receptor KDR. However, VEGF-induced tyrosine phosphorylation of p42^mapk was not further enhanced secondary to incubation of endothelial cells under hypoxic conditions, whereas tyrosine phosphorylation of the receptor KDR was. It is an interesting possibility that the KDR-dependent signal for enhanced mitogenicity under hypoxic conditions is not primarily being transduced through the p42^mapk pathway.^{43 44} One such possibility could be the activation of p70/p85 S6 kinase by a pathway independent of p21^ras, as recently suggested for transduction of a mitogenic signal through the receptor for PDGF.⁴⁵

The hypothesis that VEGF is relevant for stimulating angiogenesis in vivo is based on two findings: There is upregulation of VEGF in the hypoxic⁴ and ischemic tissue,⁵ and there is a positive effect of recombinant VEGF on collateral formation and tissue perfusion in ischemic areas such as the limb¹⁶ or the heart.^{8 9} This does not exclude the involvement of other systems that might act in a synergistic or redundant fashion. In fact, successful therapeutic angiogenesis has already been shown, eg, in the case of bFGF^{46 47 48 49 50} or PDGF-BB.⁵¹ There also is experimental evidence for a synergism between VEGF and bFGF in the induction of angiogenesis in vitro.⁵² It is an interesting possibility that bFGF is upregulating VEGF expression in synergy with hypoxia, which would suggest that bFGF acts both as a direct and as an indirect stimulator of angiogenesis.⁵³ Comparative in vivo studies are needed to estimate the individual impact of different angiogenic factors. Until now, no data have been reported on the regulation of FGF receptors under hypoxic or ischemic conditions. From our data, however, there is some experimental evidence for KDR being more specifically upregulated under hypoxic conditions compared with other receptor tyrosine kinases. In our hands, the PDGF ß-receptor expressed in PAE cells and stimulated with PDGF-BB failed to respond to hypoxia. In addition, the endothelial cell specificity of VEGF action makes VEGF a better candidate for the stimulation of therapeutic angiogenesis compared with bFGF, which also might induce neointimal formation.⁵⁴

Transient hypoxia may be of general physiological relevance to the endothelium. One may speculate that the hypoxia-induced upregulation of cell surface molecules may be most critical to molecules acting on the luminal surface of the arterial and capillary endothelium, where the ordinary oxygen tension is high and where this could be dramatically lowered under pathological conditions. Such a potential regulatory mechanism may also be true for other endothelial cell surface molecules, and it is an interesting perspective that the expression and therefore the functional regulation of such molecules may change with the actual state of perfusion. The observations described in this paper could provide an experimental basis for the provocative hypothesis that the endothelium is capable of adaptation to stress such as hypoxia by activating an intrinsic survival mechanism. In such a case, the endothelium could act as a potential stress sensor and could play in synergy with the stroma that enhances the endogenous expression of factors such as VEGF.³

The data presented in this article could serve to broaden the experimental basis for better understanding the mechanism of tumor angiogenesis and explaining the feasibility of therapeutic angiogenesis.^{16 55} It can be proposed that hypoxic-ischemic tissues respond better to VEGF stimulation than normal ones. They express an increased number of receptors, resulting in an increased response. This implies a relative selectivity of the agent VEGF for the therapeutic target and a reduction of potential side effects outside the target area, which may decrease the need for selective local application. This may be important because local drug delivery in the vessel wall remains a problem. Our findings provide a basis for the regional or even systemic application of VEGF to achieve therapeutic stimulation of angiogenesis in an ischemic area, provided that a remaining perfusion to that area is being maintained that will allow VEGF to get there.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor HUVE = human umbilical vein endothelial PAE = porcine aortic endothelial PDGF = platelet-derived growth factor PDGF-ßR = PDGF ß-receptor SSC = saline sodium citrate VEGF = vascular endothelial growth factor

	Acknowledgments

 
This work was supported in part by a grant from Fritz-Thyssen-Stiftung, Cologne, Germany (Dr Waltenberger). We wish to thank Masabumi Shibuya, Tokyo, Japan, for providing Flt-1 cDNA; Carl-Henrik Heldin, Uppsala, Sweden, for providing PDGF-BB and the R3 antiserum; and Denis Gospodarowicz, Chiron Corp, Emeryville, Calif, for the kind gift of VEGF.

Received January 11, 1996; revision received March 20, 1996; accepted April 1, 1996.

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