©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor 1 Down-regulates Vascular Endothelial Growth Factor Receptor 2/flk-1 Expression in Vascular Endothelial Cells (*)

(Received for publication, February 26, 1996)

Stefano J. Mandriota Pierre-Alain Menoud (1) Michael S. Pepper (§)

From the Department of Morphology and Division of Oncology, University Medical Center, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta1 (TGF-beta1) reduced the high affinity I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.


INTRODUCTION

The formation of new blood vessels is essential for the development and maintenance of all organ systems. During embryogenesis, endothelial cells of primordial vessels differentiate from mesodermal precursors called angioblasts (vasculogenesis). During subsequent organogenesis, new capillary blood vessels are formed either by vasculogenesis or originate as endothelial sprouts from pre-existing vessels (angiogenesis). These two processes lead to the formation and maintenance of the vascular tree. Angiogenesis is also responsible for physiological and pathological neovascularization which occurs in the adult organism. Tumor angiogenesis is the most extensively studied example. It is permissive for tumor growth, and following tumor cell intravasation into newly formed vessels is responsible for the formation of metastases (reviewed by Folkman(1995) and Risau and Flamme(1995)).

Endothelial cell tyrosine kinase receptors are of fundamental importance in the transmission of both differentiation and angiogenic signals from the extracellular environment to the endothelium. Five endothelial cell-specific tyrosine kinase receptors, each of which has a specific role in blood vessel formation, have been identified. These include Tie-1, Tie-2 (also known as Tek), Flt-1, Flt-4, and Flk-1/KDR (reviewed by Mustonen and Alitalo(1995)). While the ligands for Tie-1 and Tie-2 have not yet been identified, Flk-1/KDR and Flt-1 (which in this paper are referred to as Flk-1 and Flt, respectively) are receptors for vascular endothelial growth factor (VEGF) (^1)(de Vries et al., 1992; Terman et al., 1992). Flk-1 has been shown recently to be a receptor for VEGF-C, a novel VEGF-related endothelial cell growth factor which also binds to and induces autophosphorylation of Flt-4 (Joukov et al., 1995). The importance of the VEGF/VEGF receptor (VEGFR) system in blood vessel formation can be summarized as follows: (i) spatiotemporal expression of both VEGF and VEGFRs correlates well with phases of vasculogenesis and angiogenesis in the embryo, and with phases of neovascularization in the adult (reviewed by Mustonen and Alitalo(1995)); (ii) mice lacking Flk-1 or Flt die at early stages of development, the former being essential for endothelial cell differentiation, the latter for correct vascular assembly (Fong et al., 1995; Shalaby et al., 1995); (iii) VEGF is the major angiogenic factor in an animal model of ischemia-stimulated retinal neovascularization (Aiello et al., 1995); (iv) antibodies to VEGF, as well as a dominant-negative form of Flk-1, are able to block tumor angiogenesis and tumor growth in vivo (Kim et al., 1993; Millauer et al., 1994).

Angiogenesis is believed to proceed in at least two phases: (i) the activation phase, in which endothelial cells degrade their basement membrane, and form capillary ``sprouts'' by migrating and proliferating in the surrounding stroma; and (ii) the resolution phase, in which migration and proliferation cease and a new basement membrane is deposited (Pepper et al., 1996a). Both phases appear to be determined by a balance between positive and negative regulators. One of the elements likely to play a key role in this equilibrium is the regulation of expression of endothelial cell tyrosine kinase receptors. Consistent with this hypothesis, flk-1, flt, flt-4, tie-1, and tie-2 expression correlates well with phases of capillary growth, although in a few settings high expression of tyrosine kinase receptors persists after endothelial cell proliferation and migration have ceased (Mustonen and Alitalo, 1995; Pepper et al., 1996a). Although VEGF receptors (Matthews et al., 1991; Shibuya et al., 1990) have been characterized extensively at the level of expression (Mustonen and Alitalo, 1995), high affinity VEGF binding (de Vries et al., 1992; Terman et al., 1992), phosphorylation, and other signal transduction properties (Waltenberger et al., 1994), very little is known about factors which regulate expression in vascular endothelial cells. Hypoxia, which up-regulates VEGF expression in a variety of cell types (Brogi et al., 1994; Namiki et al., 1995; Shweiki et al., 1992), has been reported recently to induce the release of a factor from myoblasts and smooth muscle cells which increases expression of flk-1 (Brogi et al., 1996). The identity of this factor remains to be determined.

We have cloned partial cDNAs encoding bovine flk-1 and flt and used them as probes to study VEGFR expression in bovine microvascular and large vessel (aortic) endothelial cells. Both cell lines express flk-1, but not flt. Incubation of the cells with the multifunctional angiogenic cytokine transforming growth factor beta1 (TGF-beta1) (Pepper et al., 1996a) results in a rapid and marked decrease in flk-1 expression at levels of mRNA, total protein, and cell surface I-VEGF binding capacity.


EXPERIMENTAL PROCEDURES

Molecular Cloning of Bovine flk-1 and flt Partial cDNAs

For flk-1, degenerate primers were designed from the conserved amino acid sequences QRDLDWLW and MISYAGMV, located in the first two IgG-like domains of human and mouse Flk-1 (Matthews et al., 1991; Terman et al., 1991). The primer sequences used were: 5`-A(A/G)(A/C)GNGA(C/T)(C/T)TNGA(C/T)TGG(C/T)TNTGG and 5`-ACCATNCCNGC(A/G)TAN(C/G)(A/T)(A/G/T)ATCAT. An 400-bp RT-PCR product was amplified from 2 µg of bovine microvascular endothelial (BME) cell total RNA (Macfarlane and Dahle, 1993) and cloned into the SmaI site of pBluescript KS (Stratagene). For flt, two pairs of nested primers were designed from the conserved amino acid sequences KDAALHM and CCSPPPD (outer pair), or RLPLKWM and MSLERIK (inner pair), in the intracellular domain of human, mouse, and rat Flt (Finnerty et al., 1993; Shibuya et al., 1990; Yamane et al., 1994). The primer sequences used were: 5`-ACAAGGA(C/T)GCAGCN(C/T)TNCA(C/T)ATG and 5`- TCNGGNGGNGGN(C/G)(A/T)(A/G)CA(A/G)CA (outer pair); 5`- CCGGAATTCGACTTCC(T/C)CT(G/A)AAATGGATG and 5`-CGCGGATCCTTTGATTCTTTCCAGGCTCAT (inner pair). Starting from 2 µg of bovine adult kidney total RNA, an 500-bp RT-PCR product, resulting from the second cycle of amplification with the inner pair of primers, was purified, digested with EcoRI and BamHI (which are restriction sites included in the 5` end of the primers), and subcloned into the corresponding sites of pBluescript KS. Cloned PCR products were sequenced on both strands by the chain termination method using successive primers (Sanger et al., 1977).

Cell Culture

Adrenal cortex-derived BME cells, kindly provided by Drs. M. B. Furie and S. C. Silverstein (Columbia University, New York) (Furie et al., 1984), and bovine aortic endothelial (BAE) cells (Pepper et al., 1992) were cultured as described (Mandriota et al., 1995). BME cells were used between passages 18 and 22, and BAE cells between passages 10 and 17.

Radioiodination of VEGF

Recombinant human VEGF (165-amino acid species; PeproTech Inc., Rocky Hill, NJ), iodinated by the IODOGEN (Pierce) method (Vassalli et al. 1984) using NaI (Amersham International plc, Little Chalfont, Buckinghamshire, UK) to a specific activity of 3.0-4.5 times 10^6 cpm/µg, was employed for cell binding experiments. VEGF iodinated by the chloramine T method (Vaisman et al., 1990) to a specific activity of 5.0 times 10^7 cpm/µg was used for cross-linking experiments.

Cell Binding of I-VEGF

36 h after the last medium change, TGF-beta1 (R& Systems, Minneapolis, MN) was added at the indicated concentrations to the culture medium of confluent endothelial cell monolayers in 23-mm tissue culture wells. After a 15-h incubation, cells were acid-treated and tested for their capacity to bind I-VEGF (at a concentration of 150 pM) as described (Mandriota et al., 1995). Nonspecific binding was determined by adding a 50-fold molar excess of unlabeled VEGF to the binding medium of parallel cultures and was subtracted from all samples. Samples were assayed in duplicate.

Cross-linking of I-VEGF to Cells

Confluent BAE cell monolayers in 60-mm tissue culture dishes were incubated in the presence of 1 ng/ml TGF-beta1, 30 ng/ml VEGF, or growth medium alone. At the end of the incubation, I-VEGF (at a concentration of 500 pM) was bound and subsequently cross-linked to acid-treated cells by dissuccinimidyl suberate (DSS; Pierce) as described (Mandriota et al., 1995), except that DSS was used at a concentration of 0.15 mM for 20 min at 4 °C. Cells were washed four times with ice-cold phosphate-buffered saline and lysed in 50 µl/dish TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100) containing 200 Kunitz inhibitory units/ml of Trasylol, 1 µg/ml pepstatin A, and 2 µg/ml antipain. Total protein concentration in cell lysates was measured with the BCA protein assay kit (Pierce). 150 µg of total protein of each sample were electrophoresed in an SDS-5% polyacrylamide gel under reducing conditions. Gels were dried and exposed to Kodak XAR-5 films (Eastman Kodak Co.) at -80 °C.

Metabolic Labeling of BME Cells and Immunoprecipitation

36 h after the last medium change, confluent BME cell monolayers in 60-mm tissue culture dishes were washed twice with minimal essential medium without cysteine and methionine (Life Technologies, Paisley, Scotland) and incubated in the same medium for 20 min at 37 °C. Medium was removed and the cells were incubated for 8 h at 37 °C in the presence of minimal essential medium without cysteine and methionine supplemented with 2% donor calf serum (Flow Laboratories, Baar, Switzerland) and 60 µCi/ml of Pro-mixL-S in vivo cell labeling mix (Amersham), with or without 1 ng/ml TGF-beta1. At the end of the incubation, cells were washed four times with PBS and lysed in TBST containing proteinase inhibitors as described above. Total cell lysates (1.5 times 10^6 cpm/sample) were immunoprecipitated with a polyclonal antibody (C-1158, Santa Cruz Biotechnology Inc., Santa Cruz, CA) raised against a peptide mapping to the carboxyl terminus of mouse Flk-1 (amino acids 1158-1345), according to the manufacturer's instructions. Immunoprecipitates were run in an SDS-5% polyacrylamide gel under reducing conditions. Gels were fixed for 30 min in 10% acetic acid, 30% ethanol, soaked for 30 min in Amplify (Amersham), dried, and exposed to Kodak XAR-5 films.

Immunoprecipitation/Western Blotting

Confluent BAE cell monolayers in 15-cm tissue culture dishes were incubated with 1 ng/ml TGF-beta1 or with culture medium alone. Cells were lysed at various time points, and 3 mg of total cell protein were immunoprecipitated using the antibody C-1158 as described above. Immunoprecipitates were electrophoresed as described above and transferred to a polyvinylidene difluoride membrane (PVDF, Amersham) by electroblotting (Sambrook et al., 1989). Flk-1 was revealed by means of the ECL immunodetection system (Amersham) using the polyclonal antibody C-20 (Santa Cruz Biotechnology), according to the manufacturer's instructions. The C-20 antibody recognizes the mouse Flk-1 carboxyl-terminal amino acids 1348-1367.

RNA Purification, Northern Blot Analysis, and RNase Protection Assay

TGF-beta1 was added to the culture medium of confluent endothelial cell monolayers 36 h after the last medium change. At the end of the incubation, total cellular RNA was purified as described (Chomczynski and Sacchi, 1987). In vitro transcription of P-labeled cRNA probes from pBS-bflk-1, pBS-bflt, and pSP65-hFGFR-1 (which contains a 700-bp EcoRI fragment of the human fibroblast growth factor receptor-1 (FGFR-1) cDNA; Isacchi et al. (1990)), Northern blot analysis and RNase protection assay were as described (Pepper et al. 1990, 1993a, 1995).

Densitometry

Autoradiograms were scanned with a Laser ScanJet IIcx instrument (Hewlett Packard Co., Boise, ID) and bands were quantitated using Image 1.5 (National Center for Supercomputing Applications, Urbana-Champaign, IL).


RESULTS

To identify VEGF receptor(s) expressed by BME and BAE cells, partial cDNAs for bovine flk-1 and flt were cloned by RT-PCR using degenerate primers. 400-bp (flk-1) and 500-bp (flt) fragments were cloned and sequenced. As expected on the basis of the position of the primers chosen, the cloned bovine cDNA fragments showed strong sequence similarity with the first two IgG-like domains of human and mouse flk-1 (Matthews et al., 1991; Terman et al., 1991) and with the region extending between the presumptive autophosphorylation site and the translational end of human, mouse, and rat flt (Finnerty et al., 1993; Shibuya et al., 1990; Yamane et al., 1994). Identities were: (i) for bovine flk-1: 86% and 78% (nucleotides), 85% and 75% (amino acids) with human and mouse flk-1, respectively; (ii) for bovine flt: 87%, 82%. and 83% (nucleotides); 92%, 87%, and 88% (amino acids) with human, mouse, and rat flt (Fig. 1, A and B). Complementary RNA probes transcribed from both fragments were protected by adult bovine heart and kidney RNAs in an RNase protection assay, thereby confirming their complementarity to RNAs of bovine origin (Fig. 2). BME and BAE cell RNA analysis revealed that both cell lines express flk-1 but not flt (Fig. 2). flt expression was also undetectable in both BME and BAE cells by RT-PCR using the same primers described for cDNA cloning (data not shown).


Figure 1: Partial nucleic acid sequences of bovine RT-PCR-derived flk-1 (A) and flt (B) cDNAs and comparison with amino acid sequences from other species. flk-1 identity: bovine-human, 85%; bovine-mouse, 75%. A higher degree of conservation is observed between bovine, human, mouse, and rat flt proteins (bovine-human, 92%; bovine-mouse, 87%; bovine-rat, 88%) (EMBL accession numbers X94298 (bflk-1) and X94263 (bflt)).




Figure 2: Ribonuclease protection assay of flk-1 and flt mRNAs in BME and BAE cells. Purified P-labeled 381-bp bovine flk-1 or 459-bp bovine flt cRNAs (probe) were hybridized to buffer (probe + h.m.), yeast tRNA (tRNA), or to 10 µg of total RNA from bovine adult heart, kidney, BME, or BAE cells. SP6, control template marker (New England Biolabs). To confirm RNA integrity, 2 µg of total RNA from the samples used in the ribonuclease protection assay were denatured by glyoxal, electrophoresed in a 1.0% agarose gel, and stained with ethidium bromide (bottom panel).



We have previously reported that TGF-beta1 acts in a biphasic manner on VEGF-induced in vitro angiogenesis. At picogram/ml concentrations, TGF-beta1 enhanced the effect of VEGF, whereas at nanogram/ml concentrations it had an inhibitory effect (Pepper et al., 1993b). To explain this phenomenon, we hypothesized that TGF-beta1 might modulate flk-1 expression in vascular endothelial cells. To explore this possibility, confluent cultures of BME or BAE cells were incubated overnight (15 h) in the presence of increasing concentrations (from 1 pg/ml to 10 ng/ml) of TGF-beta1. Cells were acid-treated (conditions known to detach growth factors from their high affinity cell surface receptors (Haigler et al., 1980)) and assayed for their capacity to bind I-VEGF with high affinity. TGF-beta1 decreased the capacity of BME and BAE cells to bind I-VEGF in a dose-dependent manner, with a 2.0- to 2.7-fold decrease at 1-10 ng/ml TGF-beta1 (Fig. 3). In parallel experiments performed under identical conditions, treatment of BME and BAE cells with 2.2-100 ng/ml VEGF resulted in a 2.2- to 10-fold reduction in their capacity to bind I-VEGF (data not shown).


Figure 3: TGF-beta1 decreases I-VEGF binding capacity of BME and BAE cells. Confluent cultures of BME or BAE cells were incubated with the indicated concentrations of TGF-beta1 or with cytokine-free medium (C) for 15 h. At the end of the incubation, I-VEGF (150 pM) was bound to acid-treated cells. Radioactivity of cell lysates from duplicate wells was measured in a -counter. A 2.0- to 2.7-fold decrease was detected in BME and BAE cells incubated with 1-10 ng/ml TGF-beta1. Nonspecific binding was determined by adding a 50-fold molar excess of cold VEGF in parallel cultures and was subtracted from all samples. No significant differences in cell numbers were observed between control and TGF-beta1-treated cells (data not shown). Typical results from a representative experiment are shown.



To further characterize the binding of I-VEGF to vascular endothelial cells, confluent cultures of BAE cells were incubated for 8 h or 15 h in the presence of 1 ng/ml TGF-beta1 or 30 ng/ml VEGF. At the end of the incubation, the cells were acid-treated and chloramine T-labeled I-VEGF was cross-linked to the cell surface by DSS. Cell lysates were electrophoresed under denaturing conditions. Following autoradiography, a prominent band corresponding in size to the expected molecular mass of the VEGF-Flk-1 complex (about 230-235 kDa) was detected (Fig. 4A). The intensity of the band was markedly reduced (3.5-fold; 8 h) or almost undetectable (15 h) in TGF-beta1-treated cells when compared to controls (Fig. 4A). While VEGF had no effect on flk-1 expression at both mRNA and total protein levels (data not shown), the 230-235-kDa band was undetectable in VEGF-treated cells (Fig. 4A), which is consistent with the cell binding results. Similar results were obtained with different ligand and DSS concentrations (100 pMI-VEGF, 0.15 mM DSS; 100 pMI-VEGF, 0.5 mM DSS; 500 pMI-VEGF, 0.5 mM DSS), as well as under nondenaturing conditions (data not shown). No bands were detectable if IODOGEN-labeled VEGF was cross-linked to the cells (data not shown), probably because of its low specific activity (see ``Experimental Procedures''). As the VEGF used in binding and cross-linking assays had a M(r) of 45,000, and BAE cells express flk-1 but not flt mRNA (see above), we concluded that TGF-beta1 down-regulates VEGF binding to a cell surface monomeric molecule indistinguishable from Flk-1 on the basis of its affinity for VEGF, molecular size (185-190 kDa), and apparent internalization after VEGF binding (Matthews et al., 1991; Millauer et al., 1993; Terman et al., 1992; Ullrich and Schlessinger, 1990; Waltenberger et al., 1994).


Figure 4: TGF-beta1 decreases cell surface and total Flk-1 protein expression in BME and BAE cells. A, confluent cultures of BAE cells were incubated with 1 ng/ml TGF-beta1 (T), 30 ng/ml VEGF (V), or cytokine-free medium (C) for the times indicated. At the end of the incubation, cells were processed as described in the legend to Fig. 3, except that I-VEGF (iodinated by chloramine T) was cross-linked to the cell surface by DSS. Cell lysates (150 µg/sample) were electrophoresed in an SDS-5% polyacrylamide gel under reducing conditions. B, confluent cultures of BME cells were labeled by S-Met/Cys and incubated for 8 h with (T) or without (C) 1 ng/ml TGF-beta1. Cell lysates (1.5 times 10^6 cpm/sample) were immunoprecipitated with a polyclonal antibody (C-1158) recognizing a peptide (amino acids 1158-1345) in the mouse Flk-1 carboxyl terminus. Immunoprecipitates were run in an SDS-5% polyacrylamide gel under reducing conditions. C, confluent cultures of BAE cells were incubated with 1 ng/ml TGF-beta1 (T) or with cytokine-free medium (C) for the times indicated. At the end of the incubation, cell lysates (3 mg/sample) were immunoprecipitated as described in B. After electrophoresis, immunoprecipitates were transferred to a PVDF membrane. Membranes were incubated with a second antibody to Flk-1 (C-20), recognizing a peptide in the mouse Flk-1 carboxyl terminus (amino acids 1348-1367) which is different from that recognized by the antibody C-1158 (used for immunoprecipitation). Flk-1/C-20 complexes were detected by the ECL immunodetection system.



The effect of TGF-beta1 on flk-1 expression was investigated in confluent cultures of BME cells radiolabeled with [S]Met/Cys and incubated for 8 h in the presence of 1 ng/ml TGF-beta1. Cell lysates were immunoprecipitated with an antibody (C-1158) recognizing a peptide mapping to the carboxyl terminus of mouse Flk-1. Analysis of immunoprecipitates by SDS-PAGE electrophoresis and autoradiography revealed a single band with an apparent molecular mass of about 200 kDa (Fig. 4B), consistent with our cross-linking results and with the published molecular mass of Flk-1 (Matthews et al., 1991; Millauer et al., 1993; Terman et al., 1992; Waltenberger et al., 1994). The intensity of the band was decreased 2.5-fold in TGF-beta1-treated cells when compared to controls (Fig. 4B), which is consistent with the cell binding results (Fig. 3). To confirm the specificity of the antibody, confluent cultures of BAE cells were incubated from 4 to 15 h with 1 ng/ml TGF-beta1. Cell lysates were immunoprecipitated using the C-1158 antibody. Immunoprecipitates were electrophoresed, blotted onto a PVDF membrane, and incubated with a second antibody (C-20) which recognizes a peptide in the carboxyl terminus of Flk-1 (amino acids 1348-1367) which is different from that recognized by the antibody C-1158 (amino acids 1158-1345). A major band of about 200 kDa and a faint band of about 170 kDa, possibly corresponding to differentially glycosylated forms of Flk-1, were detected (Fig. 4C). At all time points tested, the intensity of both bands decreased in cells treated with TGF-beta1 when compared to controls (Fig. 4C). By scanning densitometry, a 4.5-fold decrease of Flk-1 expression was detected after 15 h. No signal was detectable if the antibody used for Western blotting was preadsorbed with the corresponding peptide, thereby demonstrating the specificity of both bands (data not shown).

Finally, we determined the effect of TGF-beta1 on BME and BAE cell flk-1 mRNA expression. Confluent cultures of BME or BAE cells were incubated overnight (15 h) in the presence of increasing concentrations of TGF-beta1 (from 1 pg/ml to 10 ng/ml). Total cellular RNA was analyzed by Northern blot (BME cells, Fig. 5A) or by RNase protection (BAE cells, Fig. 5B). TGF-beta1 decreased flk-1 mRNA levels in both cell lines in a dose-dependent manner (Fig. 5, A and B). By scanning densitometry, a 2.5-fold or a 9-fold decrease were detected in BME or BAE cells treated with 1 or 3 ng/ml TGF-beta1, respectively. In contrast, TGF-beta1 had no effect on FGFR-1/flg expression by BME cells (Fig. 5A).


Figure 5: TGF-beta1 decreases flk-1 mRNA levels in BME and BAE cells. BME and BAE cells were incubated with the indicated concentrations of TGF-beta1 or with cytokine-free medium (C) for 15 h. A, replicate Northern blot filters containing 5 µg of total BME cell RNA per lane were hybridized to a P-labeled bovine flk-1 or human FGFR-1/flg cRNA probe. Methylene blue staining (bottom panel) reveals 28 S and 18 S rRNAs and demonstrates uniformity of loading and RNA integrity. B, purified P-labeled 381-bp bovine flk-1 cRNA (probe) was hybridized to buffer (probe + h.m.), yeast tRNA (tRNA), or 10 µg of total RNA from BAE cells incubated for 15 h with cytokine-free medium (C) or with the indicated concentrations of TGF-beta1. SP6, control template marker (New England Biolabs). To confirm RNA integrity, 2 µg of total RNA from the samples used in the ribonuclease protection assay were denatured by glyoxal, electrophoresed in a 1.0% agarose gel, and stained with ethidium bromide (bottom panel).




DISCUSSION

Endothelial cell activation status is determined by an equilibrium between positive and negative regulators of endothelial cell proliferation, migration, and proteinase production (Pepper et al., 1996a, 1996b). One element likely to play a central role in this equilibrium is the regulation of endothelial cell tyrosine kinase receptor expression by environmental factors. Among the well characterized endothelial cell tyrosine kinase receptors, Flt and Flk-1 are receptors for VEGF, an endothelial cell-specific mitogen whose importance in both physiological and pathological angiogenesis is well established (Mustonen and Alitalo, 1995; Pepper et al., 1996a). However, very little is known about factors which regulate VEGFR expression in vascular endothelial cells.

In the present study we report that TGF-beta1 down-regulates the expression of bovine endothelial cell Flk-1, the major signal-transducing tyrosine kinase receptor for VEGF (Waltenberger et al., 1994). We have characterized this effect at levels of (i) mRNA (by means of Northern blotting and RNase protection assay), (ii) total protein (by immunoprecipitation, alone or followed by Western blotting with an antibody directed against a different Flk-1 peptide), (iii) cell surface (by high affinity binding and cross-linking of iodinated VEGF to the cells). The down-regulation of flk-1 expression by TGF-beta1 may be responsible for the inhibitory effect of this cytokine (at nanogram/ml concentrations) on VEGF-induced in vitro angiogenesis (Pepper et al., 1993b).

The findings that VEGF increases plasminogen activator (PA) activity in vascular endothelial cells (Mandriota et al., 1995; Pepper et al., 1991) and that plasmin is able to activate latent TGF-beta1 (Flaumenhaft et al., 1992; Lyons et al., 1990; Sato et al., 1990; Sato and Rifkin, 1989) raise the possibility that a complex self-regulating mechanism of VEGF signal transduction may exist during angiogenesis. Thus, in the initial phases of angiogenesis, endothelial cells respond to VEGF by increasing PA activity, migration, and proliferation, which results in the formation of new capillary sprouts. As a consequence of increased plasmin formation, latent TGF-beta1 is activated. This in turn might decrease flk-1 expression and thereby reduce the effects of VEGF in vascular endothelial cells. This would result in endothelial cell quiescence, reduced levels of plasmin activity, and deposition of a new basement membrane, all of which are characteristic of the later stages of new vessel formation (Ausprunk and Folkman, 1977; Paku and Paweletz, 1991). Finally, as a consequence of decreased plasmin activity, TGF-beta1 would no longer be activated, flk-1 expression would increase and endothelial cells of newly formed vessels could enter another cycle of invasion in response to VEGF.

In conclusion, our results, taken together with the recent finding that Flk-1 is a receptor for VEGF-C, a novel VEGF-related endothelial growth factor which also binds to and induces autophosphorylation of Flt-4 (Joukov et al., 1995), implicate TGF-beta1 as a major regulator of VEGF/Flk-1-mediated signal transduction in vascular endothelial cells.


FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grants 3100-043364.95 and 32-39212.93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X94298 [GenBank](bflk-1) and X94263 [GenBank](bflt).

§
To whom correspondence should be addressed: Dept. of Morphology, University Medical Center, 1, rue Michel Servet, 1211 Geneva 4, Switzerland. Tel.: 0041-22-702-5291; Fax: 0041-22-347-3334. pepper{at}cmu.unige.ch.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; TGF-beta1, transforming growth factor beta1; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); BME, bovine microvascular endothelial; BAE, bovine aortic endothelial; DSS, dissuccinimidyl suberate; PVDF, polyvinylidene difluoride; PA, plasminogen activator.


ACKNOWLEDGEMENTS

We are grateful to Drs. M. B. Furie and S. C. Silverstein for the BME cells, Dr. A. Isacchi for the human FGFR-1/flg cDNA, Drs. G. Bunone, G. Collo, and J.-P. Paccaud for assistance in initial phases of this work, and Drs. R. Montesano and L. Orci for continued support. Technical assistance was provided by C. Di Sanza, E. Garrido, and M. Quayzin, and photographic work was done by B. Favri.


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