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Am J Physiol Heart Circ Physiol 279: H2116-H2123, 2000;
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Vol. 279, Issue 5, H2116-H2123, November 2000

Inhibition of adenosine kinase induces expression of VEGF mRNA and protein in myocardial myoblasts

Jian-Wei Gu1, Bruce R. Ito2, Amanda Sartin1, Nan Frascogna1, Michael Moore1, and Thomas H. Adair1

1 Angiogenesis Research Laboratories, Department of Physiology and Biophysics, Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216; and 2 Metabasis Therapeutics, Inc., San Diego, California 92121


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested whether increased endogenous adenosine produced by the adenosine kinase inhibitor GP-515 (Metabasis Therapeutics) can induce vascular endothelial growth factor (VEGF) expression in cultured rat myocardial myoblasts (RMMs). RMMs were cultured for 18 h in the absence (control) and presence of GP-515, adenosine (Ado), adenosine deaminase (ADA), or GP-515 + ADA. GP-515 (0.2-200 µM) caused a dose-related increase in VEGF protein expression (1.99-2.84 ng/mg total cell protein); control VEGF was 1.84 ± 0.05 ng/mg. GP-515 at 2 and 20 µM also increased VEGF mRNA by 1.67- and 1.82-fold, respectively. ADA (10 U/ml) decreased baseline VEGF protein levels by 60% and completely blocked GP-515 induction of VEGF. Ado (20 µM) and GP-515 (20 µM) caused a 59 and 39% increase in VEGF protein expression and a 98 and 33% increase in human umbilical vein endothelial cell proliferation, respectively, after 24 h of exposure. GP-515 (20 µM) had no effect on VEGF protein expression during severe hypoxia (1% O2) but increased VEGF by an additional 27% during mild hypoxia (10% O2). These results indicate that raising endogenous levels of Ado through inhibition of adenosine kinase can increase the expression of VEGF and stimulate endothelial cell proliferation during normoxic and hypoxic conditions.

endothelial cells; adenosine deaminase; vascular maintenance factor; adenosine receptors; growth factors; angiogenesis; GP-515; vascular endothelial growth factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that adenosine is an important physiological regulator of myocardial blood flow (5, 17, 34). When the O2 supply-to-demand ratio decreases, adenosine formation increases, which leads to an increase in cytosolic and interstitial adenosine levels (11, 12, 43). The adenosine released under these conditions is thought to play an important role in reestablishing the balance between energy supply and demand (44) by virtue of its potent vasodilatory (45) and antiadrenergic (13, 39) effects. Adenosine may also be involved in ischemic preconditioning (7). Evidence for novel cardiovascular actions mediated by the G protein-coupled receptor family, such as A1, A2a, A2b, and A3 (8, 49), is accumulating.

Mounting evidence suggests that adenosine may be a mediator of metabolic regulation of angiogenesis (1, 18, 48), not only because adenosine formation increases in tissues during hypoxic conditions and when metabolic rate increases, but also because adenosine can stimulate endothelial cell proliferation in vitro (18, 19, 36, 37) as well as angiogenesis in a variety of in vivo models (1-3).

Angiogenesis is often observed in hypoxic tissues as well as tissues in which the metabolic rate has increased. A growing belief is that vascular endothelial growth factor (VEGF) is a regulator of angiogenesis in physiological and pathological conditions (21, 24). It is well established that adenosine, mediated by way of adenosine A2 receptors (23, 27, 46), can upregulate VEGF mRNA and protein expression. Recent studies from this laboratory (27) have shown that endogenous adenosine can account for the majority of basal VEGF mRNA and protein expression in cultured myocardial vascular smooth muscle cells under normoxic conditions. This latter finding supports the hypothesis that adenosine could be a physiological maintenance factor for the vasculature (27).

In the heart, most of the adenosine is derived as a dephosphorylation product of free cytosolic AMP by the actions of cytosolic 5'-nucleotidase, and 90% of this adenosine is rephosphorylated back to AMP by the actions of adenosine kinase (33). Hypoxia can increase the levels of myocardial free AMP as well as the release of adenosine into the coronary blood (25, 29, 31). However, the increase in cytosolic adenosine levels (10- to 30-fold) far exceeds the increase in AMP levels (2- to 4-fold) (11). This disparity between the amounts of AMP and adenosine produced under hypoxic conditions can be explained by studies which suggest that hypoxia increases the activity of 5'-nucleotidase (30, 32) and decreases the activities of adenosine kinase (30, 34) and adenosine deaminase (32).

In the present study, we use a novel adenosine kinase inhibitor, GP-515 (Metabasis Therapeutics), which has been shown to increase endogenous adenosine levels in vitro (39) and in vivo (9), to test whether increasing adenosine by adenosine kinase inhibition 1) induces the expression of VEGF protein and mRNA in cultured rat myocardial myoblasts (RMMs) and 2) stimulates the proliferation of vascular endothelial cells. The results suggest that inhibition of adenosine kinase by GP-515 can induce the expression of VEGF and stimulate the proliferation of vascular endothelial cells mediated via increases in adenosine.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Adenosine was purchased from Fujisawa (Deerfield, IL). Adenosine kinase inhibitor, GP-515 {4-amino-1-(5-amino-5-deoxy-1-beta -D-ribofuranosyl)-3-bromo-pyrazolo-[3,4-d]-pyrimidine}, was obtained from Metabasis Therapeutics (San Diego, CA). GP-515 is a very potent adenosine kinase inhibitor, with an IC50 of 4 nM on isolated human cardiac adenosine kinase, and is quite specific (IC50 > 25 µM for adenosine deaminase, IC50 > 100 µM for AMP deaminase) and does not bind to A1 or A2 receptors or to nitrobenzylthioinosine-sensitive adenosine transporters. Adenosine deaminase was obtained from Sigma Chemical (St. Louis, MO). Deoxy-[32P]CTP was obtained from Du Pont New England Nuclear (Boston, MA). Multiprime DNA labeling system was obtained from Amersham International (Amersham, UK). VEGF ELISA kits were provided by R & D Systems (Minneapolis, MN).

Cell cultures. RMMs were obtained from American Type Culture Collection (CRL-1446). The cells were seeded into sterile culture flasks at ~5 × 104 cells/cm2 and incubated at 37°C in a humidified atmosphere of 5% CO2-20% O2-75% N2. The culture medium was DMEM (Sigma Chemical) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The initial cultured cells, after having been frozen in liquid nitrogen, reached confluence in 10-14 days. The cells were used between passages 2 and 4 after having been frozen in liquid nitrogen in the experiments. When monolayers of RMMs reached ~80% confluence, standard medium was replaced with medium having 4% heat-inactivated FBS in the absence and presence of GP-515, adenosine, adenosine deaminase, and GP-515 + adenosine deaminase. Samples were taken after various periods of incubation in a normoxic environment.

Exposure to hypoxia. Experiments were performed in a water-jacketed triple-gas incubator (Queue System, Parkersburg, WV) that controls the O2 and CO2 to within ±0.1% of the set-point value. When the cells reached ~80% confluence in a normoxic environment, the standard medium was replenished with 4% heat-inactivated medium, and the RMM monolayers were exposed to a normoxic, mild hypoxic (10% O2-5% CO2-85% N2), or severe hypoxic (1% O2-5% CO2-94% N2) environment for the next 18 h in the absence and presence of GP-515 (20 µM). Cells exposed to hypoxia still excluded trypan blue dye (>95%) and showed no morphological changes by light microscopy, and the levels of lactate dehydrogenase (LD-L 20 assay kit, Sigma Chemical) were not increased in the media.

Measurement of VEGF protein. The amount of VEGF protein was measured in the media of cultured RMMs by use of sandwich ELISA (R & D Systems). In these assays, the angiogenic growth factor in the test sample is sandwiched between a monoclonal antibody against mouse recombinant VEGF. A second polyclonal antibody against the growth factor is conjugated to horseradish peroxidase and then added to the mixture. Color develops by addition of hydrogen peroxide and chromogen tetramethylbenzidine, and the intensity is measured at 450 nm. VEGF protein levels were normalized to the total amount of cellular protein and expressed as picograms per milligram of total cell protein. Cell protein content was determined in duplicate with BSA as the standard (Bio-Rad Protein Assay Kit).

Northern blot analysis. Total RNA was prepared using a total RNA isolation kit (catalog no. 1910, Ambion, Austin, TX). Total RNA (20 µg) was electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde in 1× MOPS buffer. The gel was stained with ethidium bromide to visualize rRNA, and the RNA was transferred to nitrocellulose paper (Schleicher & Schuell, Keene, NH) overnight by capillary action in 20× saline-sodium citrate (SSC: 3 M NaCl, 0.3 M sodium citrate · 2H2O, pH 7.0). After transfer, the nitrocellulose paper was baked at 80°C under vacuum for 2 h and then prehybridized for 3 h at 42°C in a buffer containing 25 mM KPO4 (pH 7.4), 6× SSC, 5× Denhardt's solution, 50% formamide, and 50 µg/ml salmon sperm DNA. Dextran (10% final concentration) was then added to the prehybridization solution. cDNA probes were denatured by boiling and added to the hybridization solution. After hybridization overnight at 42°C, the blots were washed at 42°C in 0.2× SSC-0.1% SDS until the background radioactivity was removed.

The VEGF cDNA probe is a 580-bp EcoR I-BamH I fragment of the murine VEGF cDNA cloned into pBluescript plasmid [kindly provided by Dr. Werner Risau (42), Max-Planck Institute]. The cDNA probe was labeled with deoxy-[alpha -32P]CTP (New England Nuclear) using a multiprime random-primed DNA labeling kit (Amersham). Quantification of VEGF mRNA expression was performed on phosphor images of blots collected using a PhosphorImager (Molecular Dynamics) with ImageQuant software (version 3.3, Molecular Dynamics). To verify the relative amount of total RNA, filters were hybridized with a 32P-labeled 28S rRNA in each sample.

Cell proliferation. Cell proliferation was estimated by cell number and [3H]thymidine incorporation. Cell number was determined using a hemocytometer. The uptake of [3H]thymidine by RMMs and human umbilical vein endothelial cells (HUVECs) was used as an indicator of DNA synthesis, as described previously (26). Briefly, the cells were seeded into 24-well plates at 104 cells/cm2 in standard medium. On the following day, the medium was removed and replaced with fresh standard medium for 24 h. The cells were then cultured in 4% FBS-medium 199 in the absence (control) and presence of adenosine (20 µM) and GP-515 (20 µM) for 24 h. During the last 6 h of incubation, the cells were pulsed with [3H]thymidine (Amersham) by addition of 1 µCi/well. The cells were then washed, harvested, and processed for counting in a scintillation counter.

Statistical analysis. All determinations were performed in duplicate, and each experiment was repeated at least twice. Where indicated, values are means ± SD. Differences were considered statistically significant when P < 0.05 by paired t-test. All statistical calculations were performed using StatView software (BrainPower, Calabasas, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VEGF mRNA expression. Northern blot analyses were used to determine the effect of GP-515, an adenosine kinase inhibitor, on VEGF mRNA expression in RMMs cultured under normoxic conditions. GP-515 at 2 and 20 µM increased VEGF mRNA by 67 and 82%, respectively, after 18 h of treatment compared with the control group (Fig. 1; relative VEGF mRNA = l for control vs. 1.67 ± 0.11 and 1.82 ± 0.20 for 2 and 20 µM, respectively; P < 0.05 for both). These results suggest that inhibition of adenosine kinase by GP-515 can increase VEGF mRNA expression in a dose-related manner in cultured RMMs under normoxic conditions.


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  		Fig. 1.   Adenosine kinase inhibition increases vascular endothelial growth factor (VEGF) mRNA expression in cultured rat myocardial myoblasts (RMMs). GP-515 (2 and 20 µM) caused a 1.67 ± 0.11- and a 1.82 ± 0.20-fold (SD) increase in VEGF mRNA expression, respectively, after 18 h of incubation compared with control (n = 4). *P < 0.05.

VEGF protein expression. VEGF protein levels in media were measured using ELISA (R & D Systems) after RMMs were cultured under normoxic conditions in the absence and presence of GP-515, adenosine, adenosine deaminase, and GP-515 + adenosine deaminase. GP-515 added to culture media caused a dose-related increase in VEGF protein expression (Fig. 2). VEGF protein levels, expressed as nanograms per milligrams of total cell protein, increased by 8, 36, 39, and 54%, respectively, in RMMs after 18 h of exposure to 0.2, 2, 20, and 200 µM GP-515, respectively, compared with control (control VEGF protein = 1.84 ± 0.05 ng/mg).


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  		Fig. 2.   Adenosine kinase inhibition increases VEGF protein levels in media of RMMs. Cells incubated for 18 h with GP-515 (0.2, 2, 20, and 200 µM) demonstrated a dose-related increase in VEGF protein expression (1.99 ± 0.04, 2.50 ± 0.13, 2.56 ± 0.06, and 2.84 ± 0.11 ng/mg total cell protein); control VEGF was 1.84 ± 0.05 ng/mg total cell protein. Values are means ± SD from 2 independent series of experiments (n = 6). *P < 0.001.

The increases in VEGF protein concentrations in media caused by GP-515 and adenosine were also present after 2, 6, 12, and 24 h of treatment. In these experiments, small samples of media (200 µl) were taken from T-75 flasks (media volume = 15 ml) at various times after cells were exposed to fresh media in the absence (control) and presence of GP-515 (1 µM) or adenosine (1 µM). VEGF protein levels in media increased progressively in control cultures throughout the 24-h experiment (Fig. 3): 78.4 ± 14.3, 144.9 ± 54.6, 193.8 ± 59.7, 275.9 ± 71.4, and 547.0 ± 70.1 pg/ml after 0, 2, 6, 12, and 24 h of incubation, respectively. Adenosine significantly increased VEGF protein levels by 36% after only 2 h of treatment (P < 0.01, 2 h of adenosine vs. 2 h of control), whereas 12 h were required for GP-515 to significantly increase VEGF protein levels by 20% (P < 0.05, 12 h of GP-515 vs. 12 h of control). The effect of adenosine to increase VEGF protein levels in media persisted for the entire 24-h experiment. Adenosine and GP-515 caused a quantitatively similar increase in VEGF protein levels in media after 12 and 24 h of treatment. These findings suggest that GP-515 caused a time-dependent accumulation of endogenous adenosine, requiring many hours to attain a VEGF stimulatory concentration.


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  		Fig. 3.   Time-related effect of adenosine (Ado) and GP-515 on VEGF protein expression in cultured RMMs. Cells were cultured in media in the absence (control) and presence of GP-515 (1 µM) or adenosine (1 µM), and samples were taken at different periods of incubation. VEGF protein levels in media were determined by ELISA. VEGF protein levels in media increased progressively in control cultures throughout the 24-h experiment, and VEGF protein levels in media were 78 ± 14.3, 114.9 ± 54.6, 193.8 ± 59.7, 275.9 ± 71.4, and 547.0 ± 70.1 pg/ml. Adenosine increased VEGF protein levels by 36% after only 2 h of treatment compared with the 2-h control group, whereas 12 h were required for GP-515 to significantly increase VEGF protein levels by 20%. Values are means ± SD (n = 4). *P < 0.05, Ado vs. control. **P < 0.05, Ado or GP-515 vs. control.

The quantitative effects of adenosine and GP-515 on the induction of VEGF protein expression were determined after RMMs were exposed to adenosine (20 µM) or GP-515 (20 µM) for 18 h (Fig. 4). Adenosine and GP-515 caused a 59 and 39% increase in VEGF protein levels, respectively, compared with the control group (control VEGF protein = 1.84 ± 0.11 ng/mg; P < 0.05 for both).


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  		Fig. 4.   Comparison of effects adenosine and GP-515 on induction of VEGF protein expression in cultured RMMs. After cells were exposed to GP-515 (20 µM) or adenosine (20 µM) for 18 h, VEGF protein levels increased from a control value of 1.84 ± 0.11 to 2.56 ± 0.13 and 2.92 ± 0.18 ng/mg total protein, respectively. Values are means ± SD from 2 independent series of experiments (n = 6). *P < 0.05.

We also tested whether the effect of GP-515 on induction of VEGF could be blocked by adenosine deaminase, which destroys endogenous adenosine. After RMMs were exposed to adenosine deaminase (10 U/ml) and adenosine deaminase (10 U/ml) + GP-515 (2 µM) for 18 h, VEGF protein levels were decreased by 60% in both groups compared with the control group (Fig. 5; control VEGF protein = 1.84 ± 0.05 ng/mg; P < 0.01 for both). These results indicate that adenosine deaminase can block entirely the GP-515-induced expression of VEGF.


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  		Fig. 5.   Adenosine deaminase (ADA) blocks the effect of adenosine kinase inhibition (GP-515) on VEGF protein expression in cultured RMMs. After 18 h of incubation, VEGF protein levels were significantly decreased in groups treated with ADA (10 U/ml) and ADA + 2 µM GP-515 (0.74 ± 0.06 and 0.75 ± 0.05 ng/mg total protein, respectively) compared with control (1.84 ± 0.05 ng/mg total protein). Values are means ± SD from 2 independent series of experiments (n = 6). *P < 0.05.

Cell proliferation. The effect of GP-515 and adenosine on cell proliferation in RMMs and HUVECs was determined by counting cell number with a hemocytometer and by determining [3H]thymidine incorporation. GP-515 and adenosine increased the rate of cell proliferation in HUVECs by 98 and 33%, respectively, compared with the control group [(8.5 ± 4.3) × 105 cells/well, P < 0.01 for both] after the cells were exposed to adenosine (20 µM) and GP-515 (20 µM) for 24 h (Fig. 6A). However, GP-515 and adenosine had no effect on the rate of cell proliferation in RMMs after 24 h of treatment compared with the control group [(6.6 ± 1.2) × 105 cells/well, P > 0.05 for both]. In addition, GP-515 and adenosine increased the rate of [3H]thymidine incorporation in HUVECs by 82 and 73%, respectively, compared with the control group [(11.91 ± 1.48) × 103 cpm/well, P < 0.01 for both] after the cells were exposed to adenosine (20 µM) and GP-515 (20 µM) for 24 h (Fig. 6B). These results indicate that GP-515 had a greater stimulatory effect on HUVEC proliferation than adenosine.


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  		Fig. 6.   A: effect of adenosine and GP-515 on cell proliferation of RMMs and human umbilical vein endothelial cells (HUVECs). Cell proliferation was measured by counting cell number directly after cells were cultured in media in the absence (control) and presence of adenosine (20 µM) and GP-515 (20 µM) for 24 h. GP-515 and adenosine increased the rate of cell proliferation in HUVECs by 98 and 33%, respectively, compared with control [(8.5 ± 4.3) × 105 cells/well]. B: [3H]thymidine incorporation was determined after HUVECs were cultured in media in the absence (control) and presence of adenosine (20 µM) and GP-515 (20 µM) for 24 h. GP-515 and adenosine increased the rate of [3H]thymidine incorporation by 82 and 73%, respectively, compared with control [(11.9 ± 1.5) × 103 cpm/well]. Values are means ± SD (n = 8). *P < 0.05. cpm, Counts/minute.

Exposure to hypoxia. The effect of GP-515 on inducing VEGF protein expression in cultured RMMs was determined under mild and severe hypoxic conditions. Cells were cultured in the absence and presence of GP-515 (20 µM) under normoxia (20% O2), mild hypoxia (10% O2), and severe hypoxia (1% O2) for 18 h. Hypoxia alone increased VEGF protein levels by 61% (3.29 ± 0.10 ng/mg) and 197% (6.06 ± 0.12 ng/mg) when cells were exposed to 10 and 1% O2, respectively, compared with the normoxic control group (control VEGF protein = 2.04 ± 0.12 ng/mg; P > 0.05 for both). GP-515 (20 µM) caused a 37% increase (2.79 ± 0.27 ng/mg) in VEGF protein levels in media under normoxic conditions and a 27% (4.17 ± 0.16 ng/mg, P > 0.05) increase during mild hypoxia but had no effect during severe hypoxia (5.84 ± 0.13 ng/mg) compared with the O2-matched controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results show that the adenosine kinase inhibitor GP-515 caused a dose-dependent increase in the expression of VEGF protein and mRNA in RMMs cultured in a normoxic environment. The effect could be blocked entirely by adenosine deaminase, which inactivates/destroys endogenous adenosine. These findings support the hypothesis that adenosine kinase inhibition induces VEGF expression and that the effect is mediated by increasing the formation of endogenous adenosine. We previously demonstrated that exposing myocardial vascular smooth muscle cells to media containing exogenous adenosine or the adenosine A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)]ethyl adenosine caused a significant increase in the expression of VEGF protein and mRNA under normoxic conditions (27). Several other laboratories (23, 46) have confirmed that exogenous adenosine can enhance VEGF expression in a variety of cultured cells. The present study shows for the first time that increasing endogenous levels of adenosine through inhibition of adenosine kinase can enhance VEGF expression.

Intracellular concentrations of adenosine are low when tissues have normal oxygenation. In this normoxic situation, extracellular adenosine is transported rapidly to the intracellular compartment, where it is phosphorylated to AMP by adenosine kinase or deaminated to inosine by adenosine deaminase. The adenosine kinase salvage pathway is particularly important in normoxic tissue such as the heart, where it has been estimated that >80% of adenosine formed from AMP is rephosphorylated by adenosine kinase (33). Endogenous levels of adenosine are increased by 10- to 30-fold (11) during ischemic episodes in the myocardium, where it acts as a naturally protective metabolite. However, newly formed adenosine is removed very quickly from tissues by the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. The results of the present study indicate that blocking adenosine kinase with as little as 0.2 µM GP-515 causes endogenous adenosine to increase sufficiently to attain a VEGF stimulatory concentration.

Accumulating evidence suggests that VEGF plays a key role in tissue angiogenesis, particularly in the heart (6, 52). Furthermore, exogenous administration of VEGF has been shown to enhance angiogenesis and blood flow in ischemic cardiac and skeletal muscle (4, 47, 50). The activity of GP-515 to increase VEGF and stimulate endothelial cell proliferation suggests that it may have therapeutic value to induce angiogenesis in ischemic settings. It will be interesting to determine whether GP-515 can induce VEGF expression and angiogenesis in the intact animal.

The results of the present study show that adenosine was more effective than GP-515 in stimulating VEGF protein expression. When the cells were treated with equimolar doses (20 µM), adenosine increased VEGF protein levels by 59%, whereas GP-515 caused a 39% increase, compared with the control group (Fig. 4). Increasing the dose of GP-515 by 10-fold to 200 µM caused VEGF protein levels to increase by only 54% (Fig. 2), which is still below the level attained by 20 µM adenosine. These findings are not surprising and are likely heavily influenced by the media volume and cell number, which would impact on the kinetics and steady-state levels of endogenously produced adenosine.

We can speculate that the level of enhancement of VEGF expression caused by adenosine kinase inhibition is dependent on the accumulation of adenosine in the tissues. Several studies (35, 38, 51) have shown that adenosine at low concentrations (<5 µM for mammalian tissues) is mainly phosphorylated to AMP by adenosine kinase; however, deamination to inosine by adenosine deaminase predominates at higher concentrations. These findings are consistent with the properties of the two enzymes, because the Michaelis-Menten constant of the deaminase is considerably higher than that of the kinase (41). Therefore, the ability of adenosine kinase inhibition to increase endogenous levels of adenosine and thus to stimulate VEGF protein expression could be limited by the actions of adenosine deaminase.

This latter contention is supported by the work of Decking and associates (10). These investigators found that adenosine kinase inhibition (iodotubercidin) alone induced a 7-fold increase in adenosine release in normoxic guinea pig hearts, whereas adenosine deaminase inhibition [erythro-9-(2-hydroxy-3-nonyl)adenosine] alone induced a 2.5-fold increase in adenosine release. However, combined blockade of adenosine kinase and adenosine deaminase in the same hearts increased the release of adenosine by ~20-fold. On the basis of these findings, we hypothesize that simultaneous inhibition of adenosine kinase and adenosine deaminase would act synergistically to enhance VEGF expression and, therefore, to stimulate angiogenesis.

The present study also demonstrates that exogenous adenosine and adenosine kinase inhibition stimulate HUVEC proliferation but not RMM proliferation (Fig. 6). In other experiments, we found that neither exogenous adenosine nor adenosine kinase inhibition affected the proliferation of cultured dog coronary artery smooth muscle cells (data not published). Several other investigators have demonstrated that exogenous adenosine increases cell proliferation in cultures of human (18, 19) and bovine endothelial cells (36, 37) but not vascular smooth muscle cells (14, 15).

Recent studies have shown that adenosine, mediated by way of adenosine A2 receptors (23, 27, 46), can upregulate VEGF mRNA and protein expression. VEGF is a specific mitogen only for endothelial cells, because its receptors are restricted to endothelial cells. Upregulation of VEGF expression by adenosine could therefore explain why adenosine stimulates endothelial cell proliferation but not the proliferation of other cell types. However, we cannot exclude the possibility that adenosine might induce endothelial cell proliferation by mechanisms independent of VEGF. Ethier and Dobson (19) reported that adenosine increases DNA synthesis in HUVECs through a pathway not mediated by the adenosine A1, A2, or A3 cell surface receptors. The results of the present study indicate that GP-515 causes a greater increase in DNA synthesis and proliferation of HUVECs than exogenous adenosine (Fig. 6). The explanation of this result is not clear given the observation that, on an equimolar basis, adenosine was more effective than GP-515 in stimulating VEGF production.

We can speculate that adenosine kinase inhibition raises intracellular levels of adenosine to a greater extent than can be achieved by exogenous adenosine. This possibility is supported by the work of Decking and associates (10), which demonstrates that 90% of adenosine formed from AMP in the normoxic heart is rephosphorylated intracellularly to AMP by adenosine kinase. Although the majority of adenosine effects are mediated through cell surface receptors, there is evidence that high concentrations of adenosine can inhibit adenyl cyclase directly through an intracellular "P-site" (8). It is thus possible that this latter mechanism is in part responsible for the greater effect of adenosine kinase than exogenous adenosine inhibition on endothelial cell proliferation.

The interstitial concentration of adenosine can increase from a normal value on the order of ~100 nM (40) to as high as 40 µM (28) during hypoxic conditions. Many groups have demonstrated that physiological concentrations of adenosine can stimulate endothelial cell proliferation in vitro (18, 19, 37) as well as VEGF expression in a variety of cell cultures (23, 27, 46). Our previous studies (27) have shown that endogenous adenosine can account for the majority of basal VEGF mRNA and protein expression in cultured myocardial vascular smooth muscle cells under normoxic conditions. In the present study, we have found that adenosine deaminase decreased basal levels of VEGF protein by >60% in RMMs (Fig. 5), again supporting the notion that the majority of VEGF produced by cells under normoxic conditions is mediated by endogenous sources of adenosine. These findings are consistent with the supposition that adenosine could be important for maintaining the integrity of the endothelial cell monolayer (19) and, more importantly, that adenosine could be a physiological maintenance factor for the vasculature, as discussed in more detail elsewhere (27). Adenosine deaminase incubation also abolished the effect of GP-515 to stimulate VEGF expression, strongly implicating the role of adenosine in mediating the effect of adenosine kinase inhibition.

Adenosine kinase inhibition did not augment hypoxia-induced VEGF expression when the hypoxia was severe, but GP-515 had an additive effect on stimulating VEGF expression when combined with mild hypoxia at 10% O2. These results are consistent with previous studies from this laboratory (27) showing that severe hypoxia at 1% O2 caused a maximum increase in VEGF protein expression that could not be augmented by simultaneous treatment with adenosine. However, the adenosine A2-receptor antagonist 8-(3-chlorostyryl)-caffeine decreased the hypoxic induction of VEGF protein by 25%, suggesting that adenosine may account for ~25% of the hypoxic induction of VEGF protein when the hypoxia is severe (27). The fact that adenosine kinase inhibition could increase VEGF protein expression during mild hypoxia (Fig. 7) is consistent with the notion that GP-515 may have therapeutic value as a promotor of angiogenesis under hypoxic conditions.


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  		Fig. 7.   Effects of GP-515 on induction of VEGF protein expression in cultured RMMs under normoxic, mild hypoxic, and severe hypoxic conditions. Cells were cultured in the absence and presence of GP-515 (20 µM) under normoxia (20% O2), mild hypoxia (10% O2), and severe hypoxia (1% O2) for 18 h. VEGF levels were determined using ELISA. Hypoxia caused a concentration-related increase in VEGF protein levels in the absence of GP-515. GP-515 caused a 37% increase in VEGF protein levels in media under normoxic conditions and a 27% increase during mild hypoxia but had no effect during severe hypoxia. Values are means ± SD from 2 independent series of experiments (n = 4). *P < 0.05.

In conclusion, we have demonstrated that the adenosine kinase inhibitor GP-515 caused a dose-dependent increase in the expression of VEGF protein and mRNA in RMMs cultured in a normoxic environment. The effect could be blocked entirely by adenosine deaminase, suggesting that the effects of GP-515 on VEGF expression were, in fact, mediated by adenosine from endogenous sources; adenosine deaminase also decreased basal levels of VEGF protein by >60% in RMMs. Although the VEGF stimulatory effects of GP-515 were quantitatively small compared with that of exogenous adenosine, GP-515 caused a greater increase in DNA synthesis and proliferation of HUVECs. Other studies showed that GP-515 did not induce VEGF protein expression during severe hypoxia but had an additive effect on inducing VEGF protein levels during mild hypoxia. These findings suggest that 1) adenosine kinase inhibitors such as GP-515 might be used as therapeutic agents to increase VEGF protein levels as well as to increase the proliferation of vascular endothelial cells and stimulate angiogenesis and 2) adenosine could be an important physiological maintenance factor for the vasculature.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-51971 and American Heart Association, Mississippi Affiliate, Grant 9810181MS. J.-W. Gu was a recipient of a grant-in-aid award from the American Heart Association, Mississippi Affiliate.


    FOOTNOTES

Address for reprint requests and other correspondence: T. H. Adair, Dept. of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505 (E-mail: tadair{at}physiology.umsmed.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 October 1999; accepted in final form 25 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 279(5):H2116-H2123
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