©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of Vascular Endothelial Growth Factor Gene Expression by Interleukin-1 in Rat Aortic Smooth Muscle Cells (*)

(Received for publication, August 17, 1994; and in revised form, October 16, 1994)

Jian Li (§) Mark A. Perrella(§) (1) (2) Jer-Chia Tsai Shaw-Fang Yet Chung-Ming Hsieh Masao Yoshizumi (1) Cam Patterson (¶) Wilson O. Endege Fen Zhou Mu-En Lee (1)(**)

From the  (1)Cardiovascular Biology Laboratory, Harvard School of Public Health, the Department of Medicine, Harvard Medical School and the (2)Pulmonary andCardiovascular Divisions, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1beta (IL-1beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1beta to see if IL-1beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1beta, and IL-1beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1beta induces VEGF gene expression in smooth muscle cells. This IL-1beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.


INTRODUCTION

Neovascularization is frequently observed in human atheromatous plaques(1, 2, 3, 4, 5) , and it has been implicated in the progression of atherosclerotic lesions(5, 6) . In addition, neovascularization has been associated with hemorrhage into atheromatous plaques, which can lead to unstable angina and myocardial infarction. The fact that human atherosclerotic plaques stimulate angiogenesis (7) indicates the presence of angiogenic factors in these plaques. Although several molecules have been shown to be important in angiogenesis, vascular endothelial growth factor (VEGF) (^1)stands out because it is a direct and specific mitogen for vascular endothelial cells(8, 9) . Studies indicate that the FLT-1 and the KDR/FLK-1 receptor tyrosine kinases function as endothelial cell-specific receptors for VEGF(10, 11, 12) , and VEGF, together with its receptors, appears to function as the key regulator of physiologic (11) as well as pathologic angiogenesis(13, 14) . For example, inhibition of angiogenesis by a monoclonal antibody to VEGF (15) or by a dominant-negative FLK-1 (which antagonizes FLK-1) (16) suppresses tumor growth in vivo.

The mRNAs of several isoforms of the VEGF family (VEGF, VEGF, VEGF, and VEGF, containing 121, 165, 189, and 206 amino acids, respectively) are generated by alternative splicing from the same gene(17, 18, 19, 20) . VEGF is the predominant isoform secreted by a variety of normal and transformed cells(18, 21) . Aortic smooth muscle cells express VEGF(22) ; however, it has not been known whether interleukin-1beta (IL-1beta), which is present in atherosclerotic lesions, regulates VEGF gene expression in aortic smooth muscle cells.

We designed the present study to test whether IL-1beta induces VEGF mRNA and protein in rat aortic smooth muscle cells (RASMC). We found that IL-1beta induced VEGF mRNA in a time- and dose-dependent manner and that this induction was due to a 211% increase in the rate of VEGF mRNA transcription and a 60% increase in its half-life. Additionally, the induction of VEGF mRNA by IL-1beta was associated with a 3.3-fold increase in the level of VEGF protein in RASMC. Our data suggest that IL-1beta present in atherosclerotic lesions may promote formation of new blood vessels by inducing expression of VEGF in smooth muscle cells.


EXPERIMENTAL PROCEDURES

Cell Culture

RASMC were harvested from male Sprague-Dawley rats (200-250 g) by enzymatic dissociation according to the method of Gunther et al.(23) . The cells were cultured in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS) and supplemented with 10% fetal calf serum (HyClone, Logan, UT), penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM HEPES (pH 7.4) (Sigma). RASMC were passaged every 4-7 days, and experiments were performed on cells three to six passages from primary culture. After the cells had grown to confluence, they were placed in quiescent medium (0.4 or 2% calf serum) 16 h before the experiments. Recombinant human IL-1beta (Collaborative Biomedical, Bedford, MA) was stored at -80 °C until use. Other cytokines and growth factors used for the multiple stimulation experiments, including murine tumor necrosis factor (TNF)-alpha (Genzyme, Cambridge, MA), human transforming growth factor-beta1 (TGF-beta1) (Collaborative Biomedical, Bedford, MA), human platelet-derived growth factor (PDGF) AA (Life Technologies, Inc.), human PDGF BB (Life Technologies, Inc.), human epidermal growth factor (EGF) (Life Technologies, Inc.), human angiotensin II (AII) (Sigma), and human endothelin (ET)-1 (Peninsula Laboratories, Belmont, CA), were also stored at -80 °C prior to use.

To promote differentiation of RASMC in vitro, we coated 10-cm cell culture dishes with 2.5 ml of Matrigel (Collaborative Research) according to the manufacturer's instruction. Cells (2 times 10^6) were evenly plated on the dishes and examined for morphological changes after 24 h(24) .

Amplification of VEGF cDNA Fragment from RASMC

A rat VEGF cDNA fragment from IL-1beta-stimulated RASMC RNA was amplified by the reverse transcription polymerase chain reaction as described(25, 26) . Primers were designed according to the published cDNA sequence(19, 20, 27) . The sequences of the forward (5`-CCTCCGAAACCATGAACT-3`) and reverse (5`-TCCTTCCTCGGAGGAGT-3`) primers were used to amplify a 618-base pair fragment. The polymerase chain reaction fragment was then subcloned and sequenced by the dideoxy chain termination method to confirm its authenticity(25) .

RNA Blot Hybridization

Total RNA was obtained from cultured cells by guanidinium isothiocyanate extraction and centrifuged through cesium chloride(25) . The RNA was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters. The filters were hybridized at 68 °C for 2 h with a random-primed, P-labeled VEGF cDNA probe in QuikHyb solution (Stratagene, La Jolla, CA). The hybridized filters were then washed in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate solution at 55 °C and autoradiographed with Kodak XAR film at -80 °C for 16-24 h or stored on phosphor screens for 6-8 h. The filters were washed in a 50% formamide solution at 80 °C and rehybridized with a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to correct for differences in RNA loading(28) . An oligonucleotide probe complementary to 18 S ribosomal RNA was used to correct for loading differences in the half-life experiments (Fig. 4A) and in the experiments assessing multiple cytokine stimulation (Fig. 3). GAPDH was not used to assess total RNA loading because cytokines, in particular PDGF BB, appear to up-regulate GAPDH mRNA levels. The filters were then scanned and radioactivity was measured on a PhosphorImager running the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


Figure 4: Effect of IL-1beta on VEGF mRNA half-life and rate of transcription in RASMC. A, RASMC were exposed to vehicle (control, open circles) or IL-1beta (10 ng/ml, filled circles) for 48 h before the addition of actinomycin D (ACD, 5 µg/ml). Total RNA was extracted from the cells at the indicated times after actinomycin D administration. Northern blot analyses were performed with 10 µg of total RNA/lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to P-labeled rat VEGF and 18 S probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the VEGF probe was divided by that hybridized to the 18 S probe. The corrected density was then plotted as a percentage of the 0-h value against time (in log scale). B, confluent RASMC were not stimulated (control) or stimulated with IL-1beta for 48 h. Nuclei were isolated, and in vitro transcription was allowed to resume in the presence of [alpha-P]UTP. Equal amounts of P-labeled, in vitro-transcribed RNA probes from each group were hybridized to 1 µg of denatured VEGF and beta-actin cDNA that had been immobilized on nitrocellulose filters.




Figure 3: Response of VEGF mRNA to multiple cytokine stimulation and effect of IL-1beta on differentiated RASMC. A, RASMC were treated with vehicle (control), IL-1beta (10 ng/ml), TNF-alpha (100 ng/ml), TGF-beta1 (10 ng/ml), PDGF AA (20 ng/ml), PDGF BB (20 ng/ml), EGF (10M), AII (10M), and ET-1 (10M). Total RNA was extracted from the cells after 24 h of stimulation. Northern blot analyses were performed with 10 µg of total RNA/lane. B, RASMC were treated with vehicle (control) or IL-1beta (10 ng/ml) in the presence of Matrigel. After 24 h of stimulation, total RNA was extracted from the cells. Northern blot analyses were performed with 5 µg of total RNA/lane. After electrophoresis (A and B) the RNA was transferred to nitrocellulose filters, which were hybridized to P-labeled rat VEGF. To assess for differences in loading, the filters were also hybridized to an 18 S oligonucleotide probe.



Nuclear Run-on Analysis

Confluent RASMC were either not stimulated (control) or stimulated with IL-1beta for 48 h. The cells were subsequently lysed, and nuclei were isolated as described by Perrella et al.(29) . Nuclear suspension (200 µl) was incubated with 0.5 mM each of CTP, ATP, and GTP and with 250 µCi of P-labeled UTP (3,000 Ci/mmol, DuPont NEN). The samples were extracted with phenol/chloroform, precipitated, and resuspended at equal counts/min/ml in hybridization buffer (2.7 times 10^6 counts/min/ml). Denatured probes (1 µg) dot-blotted on nitrocellulose filters were hybridized at 40 °C for 4 days in the presence of formamide. cDNAs for the VEGF and beta-actin genes were used as probes. The filters were scanned and radioactivity was measured on a PhosphorImager running the ImageQuant software. The amount of sample hybridizing to the VEGF probe was divided by that hybridizing to the beta-actin probe, and the corrected density was reported as a percentage increase from control.

Western Blot Analysis

Whole-cell lysates were prepared from cultured smooth muscle cells not stimulated (control) or stimulated with IL-1beta (10 ng/ml) for 48 h by boiling in lysis buffer containing 1% SDS and 10 mM Tris, pH 7.4. Protein content in the smooth muscle cells was determined according to the method of Lowry as modified for the DC protein assay (Bio-Rad). Twenty µg of total protein from IL-1beta-stimulated cells and control cells was fractionated by 10% SDS-polyacrylamide gel electrophoresis, and the protein was transferred to a polyvinylidene difluoride membrane (Immobilon-P membrane, Millipore, Bedford, MA). The membrane was incubated with 5% skim milk in Tris-buffered saline for 30 min to block nonspecific absorption. VEGF was subsequently detected by incubating the membrane with a 1:500 dilution of polyclonal anti-human VEGF antibody (Santa Cruz Biotech, Santa Cruz, CA) for 2 h at room temperature followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000) for 1 h. Immune complexes were visualized using the ECL detection system (DuPont NEN). The immunoblot bands were measured by densitometric analysis of the autoradiograph.


RESULTS

Induction by IL-1beta of VEGF mRNA in RASMC

We detected a single, 3.7-kilobase VEGF message in RNA prepared from RASMC. Although the base-line level of VEGF mRNA was low, the level increased in response to IL-1beta (10 ng/ml) within 6 h and reached a maximum (a 4-fold increase) by 48 h (Fig. 1). The VEGF mRNA level declined to 2-fold that of control at 72 h (data not shown). IL-1beta also increased VEGF mRNA levels in a dose-dependent fashion (Fig. 2). As little as 0.1 ng/ml IL-1beta was able to increase the VEGF mRNA level in RASMC by 2-fold. IL-1beta at 10 ng/ml caused maximal stimulation. At a higher dose of IL-1beta (20 ng/ml), the increase in VEGF message level was not as marked. RASMC were also stimulated with a variety of cytokines and growth factors to determine the specificity of VEGF mRNA induction (Fig. 3A). The level of VEGF mRNA increased in RASMC stimulated with IL-1beta or TGF-beta1. Other cytokines, including TNF-alpha, PDGF AA, PDGF BB, EGF, AII, and ET-1, did not increase VEGF mRNA after 24 h of stimulation. Stimulating the RASMC with IL-1beta and TGF-beta1 together produced a further increase in VEGF mRNA levels, in an additive fashion (data not shown).


Figure 1: Time course of VEGF mRNA induction by IL-1beta in RASMC. RASMC were treated with IL-1beta (10 ng/ml), and total RNA was extracted from the cells at the indicated times. RNA was also extracted from control cells (control), which received vehicle (Dulbecco's phosphate-buffered saline) but not IL-1beta. Northern blot analyses were performed with 10 µg of total RNA/lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to a P-labeled RASMC VEGF probe. The filters were also hybridized with a GAPDH probe to assess loading differences. The corrected density was then plotted as a percentage of control.




Figure 2: Dose-response of VEGF mRNA induction by IL-1beta in RASMC. RASMC were incubated for 48 h with the indicated concentrations of IL-1beta, and total RNA was extracted from the cells at the end of each incubation. See Fig. 1for details.



Passaged RASMC represent a synthetic or dedifferentiated population of cells. To determine if induction of VEGF mRNA by IL-1beta is also a property of contractile or differentiated smooth muscle cells, we performed experiments on RASMC cultured on Matrigel. Matrigel is a solubilized basement membrane matrix which has recently been used to maintain cultured vascular smooth muscle cells in a highly differentiated phenotype(24) . In the presence of Matrigel, IL-1beta also increases VEGF mRNA levels (3.5-fold) in RASMC (Fig. 3B).

IL-1beta Increased the Half-life of VEGF mRNA and the Transcriptional Rate of the VEGF Gene

To determine whether IL-1beta affected the steady-state level of VEGF mRNA by decreasing its rate of degradation, we first measured the VEGF mRNA half-life in the presence of actinomycin-D (5 µg/ml). The VEGF mRNA half-life was 2 h in the absence of IL-1beta and 3.2 h in the presence of IL-1beta (Fig. 4A). Thus, the IL-1beta-induced increase in the level of VEGF mRNA in RASMC appeared to be due in part to an increase in the stability of the mRNA. However, this 60% increase in half-life does not account for the 400% increase in the level of VEGF mRNA (Fig. 1). We then performed nuclear run-on experiments to determine the rate of VEGF gene transcription in the presence or absence of IL-1beta and compared it with the rate of transcription of the beta-actin gene. IL-1beta increased the basal rate of VEGF gene transcription (measured in PhosphorImager units) by 2.1-fold but had no effect on the rate of beta-actin transcription (Fig. 4B). Thus, the IL-1beta-induced increase in the level of VEGF mRNA in RASMC must have been due to a combined increase in the transcriptional rate of the gene and the stability of its mRNA.

IL-1beta Increased VEGF Protein Levels in RASMC

We performed Western blot analysis to determine whether the IL-1beta-induced increases in VEGF mRNA levels in RASMC were associated with increases in the level of VEGF protein. The antibody to VEGF recognized a major and a minor band, with molecular masses of 23 and 18 kDa, respectively (Fig. 5). (We confirmed the specificity of the antibody before the experiment by adsorbing it to its VEGF peptide antigen; the preadsorbed antibody recognized no immunoreactive band, not shown.) The molecular mass of the major band (Fig. 5) is similar to that of VEGF; the minor band may represent VEGF or a product of the proteolysis of VEGF(22) . IL-1beta increased the density of the major band by 3.3-fold.


Figure 5: Induction of VEGF protein by IL-1beta in RASMC. Cell lysates from control and IL-1beta-treated RASMC (48 h) were fractionated by 10% SDS-polyacrylamide gel electrophoresis. Protein on the gel was transferred to an Immobilon-P membrane. VEGF was subsequently detected by immunoblotting with a polyclonal anti-human VEGF antibody (1:500 dilution).




DISCUSSION

Neovascularization within the neointima may play an important role in the progression of atherosclerotic plaques and may cause plaque hemorrhage. Additionally, because new blood vessels express vascular cell adhesion molecule-1 they become an important site of continuous inflammatory cell recruitment into plaques(6) . Thus, a thorough understanding of the mechanisms that regulate neovascularization in atherosclerotic plaques may allow us to design strategies to modify the progression of these lesions. Human atherosclerotic plaques have been shown to stimulate angiogenesis(7) ; however, the angiogenic factors operating in these plaques have not been fully elucidated.

In this report we show that IL-1beta, which is readily detectable in atherosclerotic lesions(30) , increases VEGF mRNA in RASMC in a time- and dose-dependent manner ( Fig. 1and Fig. 2). The induction of VEGF mRNA by IL-1beta does not indicate that the VEGF gene can be induced by all cytokines. Stimulation of RASMC for 24 h with other cytokines and growth factors demonstrated a significant increase in VEGF mRNA by TGF-beta1, but not by TNF-alpha, PDGF AA, PDGF BB, EGF, AII, or ET-1 (Fig. 3A). The IL-1beta-induced increases in the level of VEGF mRNA in RASMC were apparently due both to an increase in the transcriptional rate of the gene and to an increase in the stability of its mRNA (Fig. 4). Although increases in the rate of VEGF gene transcription have been suggested as the mechanism by which agents such as phorbol esters, cyclic AMP, prostaglandins E1 and E2, and transforming growth factor-beta increase VEGF mRNA levels in osteoblasts, preadipocytes, fibroblasts, and epithelial cells(27, 31, 32, 33) , in these studies no nuclear run-on experiments were performed to assess the rate of VEGF gene transcription. To our knowledge, Fig. 4B represents the first demonstration that IL-1beta increases the transcriptional rate of the VEGF gene in RASMC. Tischer et al.(17) have shown that the promoter region of the VEGF gene contains four potential AP-1 sites. Since AP-1 sites mediate the transcriptional activation of IL-2 by IL-1beta(34) , it is possible that the IL-1beta-induced increase in VEGF gene transcription is also mediated through AP-1 sites.

IL-1beta-induced increases in VEGF mRNA are associated with similar increases in VEGF protein in RASMC (Fig. 5). Although IL-1beta has been shown to be angiogenic in vivo(35, 36) , it is not mitogenic for vascular endothelial cells in culture(37) . It is likely that the angiogenic effect of IL-1beta in vivo is mediated by the induction of VEGF in vascular smooth muscle (or other) cells; the VEGF would then act on neighboring endothelial cells to induce angiogenesis.

Our data raise the possibility that IL-1beta stimulates vascular smooth muscle cells to express VEGF. This potent angiogenic factor could then stimulate the formation of new blood vessels in atherosclerotic lesions. The new vessels, which would express adhesion molecules(6) , would subsequently attract inflammatory cells. IL-1beta released by these inflammatory cells could then induce more VEGF gene expression in the atherosclerotic lesions and initiate a vicious circle.

In summary, we have shown that IL-1beta can induce VEGF mRNA and protein in RASMC. Induction of VEGF mRNA by IL-1beta is mediated by a combined increase in the transcriptional rate of the gene and the stability of the mRNA. IL-1beta-induced VEGF gene expression in vascular smooth muscle cells may have a role in promoting neovascularization and, hence, the progression of atherosclerotic lesions.


FOOTNOTES

*
This work was supported in part by a grant from Bristol-Myers Squibb. 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.

§
Contributed equally to this work.

Supported by a National Research Service Award from the National Institutes of Health.

**
Supported by a Clinician Scientist Award and a Grant-in-Aid from the American Heart Association, with funds contributed in part by the AHA, Massachusetts Affiliate. To whom all correspondence should be addressed: Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; IL-1beta, interleukin-1beta; RASMC, rat aortic smooth muscle cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF, tumor necrosis factor; EGF, epidermal growth factor; TGF, transforming growth factor; PDGF, platelet-derived growth factor; AII, angiotensin II.


ACKNOWLEDGEMENTS

We extend our gratitude to Edgar Haber for his continued enthusiasm and support of our work. We thank Dr. Haber for his helpful suggestions and critical review of the manuscript, Bonna Ith for his technical assistance, and Thomas McVarish and Photina Ree for their editorial assistance.

Note Added in Proof-While this work was in progress, Brogi et al. (Brogi, E., Wu, T., Namiki, A., and Isner, J. M.(1994) Circulation90, 649-652) reported the induction of VEGF mRNA by TGF-beta1 in vascular smooth muscle cells.


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