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(Hypertension. 1996;27:766-773.)
© 1996 American Heart Association, Inc.

Articles

Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth

Raghvendra K. Dubey; Delbert G. Gillespie; Zaichuan Mi; Fumio Suzuki; Edwin K. Jackson

From the Center for Clinical Pharmacology, Departments of Medicine (R.K.D., D.G.G., Z.M.) and Pharmacology (E.K.J.), University of Pittsburgh (Pa) Medical Center, and Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co, Sunto-Gun, Shizuoka-Ken, Japan (F.S.).

Correspondence to Dr Raghvendra K. Dubey, Center for Clinical Pharmacology, Department of Medicine, 623 Scaife Hall, 200 Lothrop St, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582.

	Abstract

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Abstract Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum–induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([³H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [³H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A₂ adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation.

Key Words: adenosine • muscle, smooth, vascular • atherosclerosis • hyperplasia • neointima

	Introduction

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Abnormal growth of vascular SMCs is importantly associated with the pathophysiology of hypertension and atherosclerosis as well as restenosis after angioplasty and bypass surgery.^{1 2 3 4 5 6 7} The growth processes contributing to the pathological vascular structural changes, such as vascular remodeling, medial hyperplasia, and neointimal formation, involve accumulation of SMCs due to a combination of proliferation and directed migration of arterial SMCs from the media to the intima.^{1 2 3 4 5 6 7} SMC growth can be stimulated by a number of autocrine/paracrine factors present within the vessel wall and circulation,^{1 2 3 4 5 6 7} as well as by physical forces such as pressure.^{3 4} In normal blood vessels, quiescence is maintained by a balance between circulating and endothelium-derived growth inhibitors and growth promoters, which interact with each other and govern SMC growth.^{3 4 5 6 7} Disruption of the balanced generation of vasoconstrictors and vasodilators or growth promoters and growth inhibitors under pathological conditions could trigger a cascade of events leading to increased SMC proliferation and migration and neointima formation.

Most studies have investigated the possible role of circulating and endothelium-derived factors in regulating SMC growth, much less attention has been directed toward understanding the role of SMC-derived factors that can regulate growth in an autocrine fashion. In this regard, SMCs are known to produce growth promoters, such as PDGF, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, transforming growth factor-ß, and endothelin,^{1 2 3 4 5 6 7 8 9} as well as growth inhibitors, such as adenosine, nitric oxide, and prostaglandins.^{3 4 5 6 10 11 12} It has been suggested that the abnormal growth of SMCs under pathological conditions associated with vascular diseases may be due to increased generation of growth promoters, decreased production of growth inhibitors, or both.^{3 5 7 13} Growth inhibitors^{3 5 7 11 12 13} maintain homeostasis within the vessel wall,^{1 2 3 4 5 6 7} so detailed knowledge of the role of different endogenous inhibitors that regulate SMC growth and maintain homeostasis is of great clinical and therapeutic significance.

We hypothesize that adenosine may be a potential endogenous factor that is important in regulating SMC growth and maintaining vascular homeostasis. The rationale for this hypothesis is that adenosine exerts several antivaso-occlusive effects, including direct vasodilation,¹⁴ inhibition of platelet aggregation,¹⁵ diminished neutrophil adhesion to vascular endothelial cells,¹⁶ attenuation of neutrophil-induced endothelial cell damage,¹⁶ stimulation of nitric oxide release from vascular endothelial cells,^{17 18} attenuation of the sympathetic nervous system,^{19 20} and inhibition of the renin-angiotensin system.^{21 22} More importantly, we have recently shown that exogenous adenosine inhibits FCS-induced SMC growth and that SMCs synthesize substantial amounts of adenosine.^{10 23}

Although adenosine induces multiple antivaso-occlusive actions, less attention has been focused on the role of endogenous adenosine in relation to SMC growth in hypertension and atherosclerosis. Adenosine is synthesized in abundance via four distinct pathways²⁴ : the intracellular ATP pathway,²⁵ the extracellular ATP pathway,²⁶ the transmethylation pathway,²⁷ and the cAMP-adenosine pathway.^{19 20 21 28} Together, these multiple pathways can generate substantial amounts of adenosine that would maintain pharmacologically and physiologically active adenosine levels within the vasculature. Furthermore, we have recently shown that SMCs effectively metabolize exogenous cAMP into adenosine.¹⁰

The actions of adenosine are receptor mediated, including primarily A₁ and A₂ adenosine receptors,^{14 15 16 17 18 19 20 21 22 23 24 29 30 31 32 33} and are governed by the steady-state adenosine levels in the extracellular space. Elimination of adenosine from the interstitial space is mediated by facilitated transport of adenosine into the cells and also by the metabolism of adenosine to inosine by adenosine deaminase, a cytosolic enzyme that is not normally available to metabolize adenosine while adenosine resides in the extracellular compartment.³⁴ Indeed, inhibition of adenosine deaminase by EHNA has been shown to substantially enhance the effects of exogenous adenosine and increase endogenous adenosine levels.³⁵ Furthermore, administration of dipyridamole, an inhibitor of cellular nucleoside transport, elevates plasma and interstitial myocardial adenosine levels^{36 37 38} and potentiates the effects of exogenously administered adenosine. Additionally, it has been shown that antiarrhythmic effects of dipyridamole are mediated through increased generation of endogenous adenosine.³⁸

We hypothesize that continuous and substantial endogenous generation of adenosine by vascular SMCs inhibits (in an autocrine/paracrine fashion) growth of vascular SMCs and thereby plays a crucial role in reducing the risk of vaso-occlusive conditions associated with hypertension, atherosclerosis, and restenosis. This hypothesis is based on the above findings, along with the facts that (1) vascular SMCs metabolize cAMP to generate adenosine, (2) vascular SMCs express both A₁ and A₂ receptors for adenosine,^{29 39} and (3) exogenous adenosine inhibits PDGF- and FCS-induced DNA synthesis by SMCs.^{23 40}

The aim of the present study was to determine whether vascular SMCs synthesize adenosine and whether SMC-derived adenosine can effectively inhibit SMC growth. For investigation of the synthesis and antiproliferative properties of endogenous adenosine, it was necessary to prevent the metabolism of SMC-derived adenosine and to stimulate SMC proliferation. In the present study, we used EHNA and dipyridamole to maintain active levels of endogenous adenosine and selected FCS to stimulate SMC proliferation. The rationale for choosing FCS is that it is the most potent growth stimulator, containing a battery of growth factors—such as PDGF, basic fibroblast growth factor, epidermal growth factor, angiotensin II, and endothelin—that have been implicated and may be involved in the pathophysiology of several vascular abnormalities, including intimal hyperplasia, hypertension-induced medial hypertrophy, and restenosis.

	Methods

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Materials
DMEM, DMEM plus Ham's F-12 medium, Hanks' balanced salt solution, penicillin, streptomycin, 0.25% trypsin-EDTA solution, and all tissue culture ware were purchased from GIBCO Laboratories. FCS was obtained from HyClone Laboratories Inc. Adenosine, EHNA, and dipyridamole were purchased from Sigma Chemical Co. [³H]Thymidine (specific activity, 11.8 Ci/mmol) was purchased from ICN Biomedicals. KF17837 was from Kyowa Hakko Kogyo Co Ltd. All other chemicals used were of tissue culture or the best grade available.

Aortic SMC Culture
Male Sprague-Dawley rats (Charles River, Wilmington, Mass) weighing 150 to 200 g were fed standard rat chow and tap water ad libitum. Aortic SMCs were cultured as explants from the ascending thoracic aortas, obtained from ether-anesthetized rats, after a midline abdominal incision including the diaphragm and as we have described previously.¹³ SMC purity was characterized by immunofluorescence staining with smooth muscle–specific anti–smooth muscle -actin monoclonal antibodies and by morphological criteria specific for smooth muscle as described in detail previously.⁴¹ SMCs were passaged by trypsinization, and cells between passages 2 and 3 were used for DNA synthesis, SMC proliferation, and adenosine biosynthesis studies.

Growth Studies
Effects of SMC-derived adenosine on SMC growth were evaluated by studying the effects of FCS-induced DNA synthesis and change in cell number in the presence and absence of EHNA or dipyridamole, which differentially increase endogenous levels of adenosine by preventing adenosine breakdown.

DNA Synthesis
We did [³H]thymidine incorporation studies to investigate the effects of SMC-derived adenosine on FCS-induced DNA synthesis. SMCs (5x10³ cells per well) were plated in 24-well tissue culture dishes and allowed to grow for 48 hours in complete culture medium (DMEM, 13 mmol/L NaHCO₃, 25 mmol/L HEPES) containing 10% FCS for 48 hours under standard tissue culture conditions. SMC monolayers were then growth-arrested by feeding complete culture medium containing 0.25% FCS for 36 hours. Growth-arrested SMCs were treated for 20 hours with complete culture medium supplemented with 2.5% FCS and containing or lacking EHNA (10, 25, 50, or 100 µmol/L), dipyridamole (0.01 to 100 µmol/L), EHNA (10 µmol/L) plus dipyridamole (0.1 µmol/L), KF17837 (1 nmol/L), EHNA (10 µmol/L) plus KF17837 (1 nmol/L), or dipyridamole (0.1 µmol/L) plus KF17837 (1 nmol/L). After 20 hours the treatments were repeated with freshly prepared solutions but supplemented with [³H]thymidine (1 µCi/mL) for an additional 4 hours. The experiments were terminated by washing the cells twice with Dulbecco's PBS and twice with ice-cold trichloroacetic acid, and the precipitate was solubilized in 500 µL of 0.3N NaOH and 0.1% sodium dodecyl sulfate after incubation at 50°C for 2 hours. Aliquots from four wells for each treatment, with 10 mL scintillation fluid, were counted in a liquid scintillation counter. Each experiment was conducted in triplicate or quadruplicate and repeated three to four times.

In preliminary studies, we observed that SMCs rapidly metabolize adenosine and that the inhibitory effects of adenosine on cell growth decrease with the increase in cell number. To test whether the decreased inhibitory effects of adenosine were due to the catabolism of adenosine by adenosine deaminase, we studied the effects of adenosine in the presence and absence of EHNA (an adenosine deaminase inhibitor) on SMCs plated at a higher density. Briefly, SMCs (2.5x10⁴ cells per well) were plated and allowed to grow for 48 hours in the presence of complete culture medium containing 10% FCS. Subconfluent SMC monolayers were growth-arrested by feeding medium containing 0.25% FCS for 48 hours. Thymidine incorporation studies were then conducted by treating the growth-arrested cells with 2.5% FCS containing or lacking adenosine (10^-5 to 10^-3 mol/L) or adenosine (10^-5 to 10^-3 mol/L) plus EHNA (10 µmol/L) and as described above. Each experiment was conducted in quadruplicate and repeated with three separate cultures.

Cell Proliferation (Cell Number)
Trypsinized SMCs in the third passage were suspended in complete culture medium containing 10% FCS and plated in a 24-well culture dish at a density of 5x10³ cells per well. After incubation for 36 hours, the cells were fed complete culture medium containing 0.25% FCS for 48 hours to growth-arrest the cells. To study the effects of SMC-derived adenosine on FCS-induced cytokinesis, we treated growth-arrested SMCs every 24 hours for 4 days with complete culture medium containing 2.5% FCS and supplemented with or without EHNA (10, 25, 50, 100, 200 µmol/L), dipyridamole (0.01 to 10 µmol/L), adenosine (100 µmol/L), adenosine (100 µmol/L) plus EHNA (10 µmol/L), or EHNA (10 µmol/L) plus dipyridamole (0.1 µmol/L). The treatments were terminated on day 5 and cells dislodged with trypsin-EDTA, diluted in Isoton-II, and counted with a hemocytometer-calibrated Coulter counter. Aliquots from three to four wells were counted for each group, and for each aliquot greater than 10³ cells were counted. Three independent experiments were performed with separate cultures.

Adenosine Synthesis by Cultured Aortic SMCs: Effects of EHNA and Dipyridamole
We have previously shown that rat aortic SMCs metabolize exogenously applied cAMP to adenosine.¹⁰ Although this finding would suggest that the SMCs are capable of generating adenosine, it is not known whether basal synthesis of adenosine occurs in intact SMCs. To evaluate whether SMCs do synthesize adenosine endogenously, we assayed its synthesis by cultured rat aortic SMCs in the presence and absence of EHNA and dipyridamole, which differentially modulate adenosine levels in vivo.^{34 35} EHNA is a potent inhibitor of adenosine deaminase, the enzyme responsible for breaking down adenosine,^{34 35} whereas dipyridamole blocks cellular nucleoside transport and inhibits phosphodiesterase activity.^{36 37 38} Briefly, SMCs (3x passage) were plated in six-well culture dishes and grown to confluence by feeding complete culture medium containing 10% FCS. On the day of the experiment, confluent monolayers of SMCs were washed twice with Dulbecco's PBS and incubated with buffered PBS (Dulbecco's PBS, 25 mmol/L HEPES, 13 mmol/L NaHCO₃) supplemented with 2.5% FCS and containing or lacking EHNA (10 µmol/L), dipyridamole (0.1 µmol/L), adenosine (10 µmol/L), or adenosine (10 µmol/L) plus EHNA (10 µmol/L) under standard tissue culture conditions. After 4 hours of incubation, the supernatants were withdrawn and transferred into ice-cold microfuge tubes and frozen at -70°C until adenosine levels were estimated. After the collection of supernatants, the cell monolayers were inspected microscopically for intactness, and the number of cells was counted.

Adenosine Analysis
Adenosine levels in the samples were analyzed with high-performance liquid chromatography using our previously described method.²⁸ Briefly, samples were thawed and centrifuged at 10 000 rpm for 5 minutes. Supernatant (80 µL) was injected into a liquid chromatographic system (Isco pump model 2350, gradient programmer model 2360, V⁴ absorbance detector, 4.6x250-mm C18 column with 5-µm particle size, and ChemResearch Data Management System). Mobile phase A was 0.1 mol/L KH₂PO₄ (pH 6.1) and mobile phase B was 80% 0.01 mol/L KH₂PO₄ (pH 3.5) and 20% methanol. Mobile phase A was maintained at 100% for 11 minutes; a 2-minute linear gradient to 50% A was initiated; 50% A was maintained for 21 minutes; a 2-minute linear gradient back to 100% A was initiated; and 100% A was maintained for at least 24 minutes before the next sample was injected. Adenosine levels were measured as the area under the chromatographic peak, and the absolute amount in each sample was calculated from a standard curve of adenosine analyzed in parallel.

Statistics
All experiments were performed in triplicate or quadruplicate with three to four separate cultures. Data for DNA synthesis and cell number are presented as mean±SE. Statistical analysis was performed with ANOVA and paired or unpaired t test, as appropriate. A value of P<.05 was considered statistically significant.

	Results

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Effect of EHNA and Dipyridamole on Adenosine Synthesis by Cultured Rat Aortic SMCs
Adenosine levels were nondetectable in samples drawn at time zero; however, adenosine levels were significantly increased in the media of SMCs collected after 4 hours of incubation (3.5±0.32 pmol per 10⁶ cells, P<.001). Treatment of SMCs with the adenosine deaminase inhibitor EHNA significantly increased adenosine levels in the media. Adenosine levels in untreated SMCs and SMCs treated with EHNA (10 µmol/L) were 3.5±0.32 and 24.5±1.2 pmol per 10⁶ cells (P<.001), respectively, an increase of approximately 700%. In contrast to EHNA, pretreatment of SMCs with dipyridamole (0.1 µmol/L) did not result in an increase in extracellular adenosine levels (Fig 1).

View larger version (28K): [in this window] [in a new window]   Figure 1. Adenosine synthesis by cultured rat aortic SMCs. Adenosine concentrations were measured in the media of confluent monolayers of SMCs treated for 4 hours under standard tissue culture conditions with buffered PBS supplemented with 2.5% FCS and containing or lacking EHNA (10 µmol/L), dipyridamole (DIP, 0.1 µmol/L), adenosine (Ade, 10 µmol/L), or adenosine (10 µmol/L) plus EHNA (10 µmol/L). Each value represents mean±SE of three experiments conducted with separate cultures. EHNA increased adenosine levels in the medium and prevented the catabolism of exogenous adenosine. *P<.01 compared with SMCs treated with buffer; P<.01 compared with SMCs treated with exogenous adenosine or EHNA alone.

In SMCs treated for 4 hours with exogenous adenosine (10 µmol/L), only a fraction (close to basal levels) of the adenosine was recovered in the media of treated SMCs (Fig 1). Compared with SMCs treated with adenosine alone, a dramatic increase in the recovery of adenosine in the media was observed in SMCs treated with adenosine (10 µmol/L) in the presence of EHNA (10 µmol/L, Fig 1). Adenosine levels in SMCs treated with adenosine in the presence and absence of EHNA were 4.9±1.5 and 92.2±10.7 pmol per 10⁶ cells, respectively. The above observations suggest that adenosine is rapidly metabolized by adenosine deaminase. Since FCS contains the enzyme adenosine deaminase, it is possible that dipyridamole-induced levels of adenosine were metabolized to inosine in the presence of FCS.

Effect of EHNA, Dipyridamole, and Adenosine on FCS-Induced SMC Growth
DNA Synthesis
Treatment of quiescent SMCs with 2.5% FCS induced DNA synthesis (P<.001). EHNA, which elevates endogenous levels of adenosine by inhibiting adenosine deaminase, inhibited FCS-induced [³H]thymidine incorporation in a concentration-dependent manner (P<.001, Fig 2). The lowest concentration of EHNA that significantly inhibited FCS-induced DNA synthesis was 10 µmol/L. A 50% decrease in FCS (2.5%)–induced thymidine incorporation by EHNA was observed at approximately 50 µmol/L.

View larger version (19K): [in this window] [in a new window]   Figure 2. Concentration-response relationships for inhibition of 2.5% FCS-induced thymidine incorporation by EHNA and dipyridamole. Results (mean±SE) are expressed as percentage of control, defined as thymidine incorporation in the presence of 2.5% FCS (n=3 to 4 experiments, each in quadruplicate). EHNA and dipyridamole inhibited FCS-induced thymidine incorporation in a concentration-dependent manner. Thymidine incorporation in controls (100%) in four separate experiments (mean±SE of disintegrations per minute per well, n=4) was 3700±66, 8323±85, 7923±191, and 8439±20. *P<.05 compared with control.

Dipyridamole, which increases endogenous adenosine by blocking its transport,^{36 37 38} also inhibited FCS-induced DNA synthesis in a concentration-dependent manner (Fig 2, P<.001). The lowest concentration at which dipyridamole significantly inhibited thymidine incorporation was 0.01 µmol/L, and a half-maximal effect of dipyridamole on DNA synthesis was observed at 0.5 µmol/L.

Cell Proliferation (Cell Number)
Compared with SMCs treated with 0.25% FCS, treatment of growth-arrested SMCs for 4 days with 2.5% FCS induced proliferation (cell number) by 8- to 10-fold (data not shown, P>.05). EHNA, as well as dipyridamole, inhibited FCS-induced increases in cell number in a concentration-dependent manner (Fig 3, P<.01). Similar to the effects on DNA synthesis, dipyridamole was more potent than EHNA in inhibiting cell proliferation (P<.05). The lowest concentrations of dipyridamole and EHNA that inhibited cell proliferation were 0.01 and 10 µmol/L, respectively (Fig 4). Half-maximal effects of EHNA and dipyridamole on cell proliferation were observed at 50 µmol/L and 1 to 10 µmol/L, respectively (Fig 3).

View larger version (69K): [in this window] [in a new window]   Figure 3. Concentration-response relationships for inhibition of 2.5% FCS-induced SMC growth (cell number) by EHNA and dipyridamole after 4 days of treatment. Results (mean±SE) are expressed as percentage of control, defined as cell number in the presence of 2.5% FCS after 4 days of treatment (n=3 to 4 experiments, each in quadruplicate). EHNA and dipyridamole inhibited FCS-induced SMC proliferation in a concentration-dependent manner. The number of cells in controls (cells treated with 2.5% FCS) was (mean±SE, n=3) 2.7±0.2x104. *P<.05 compared with control.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of EHNA on the modulating effects of exogenous adenosine on 2.5% FCS-induced thymidine incorporation. EHNA (10 µmol/L) enhanced the inhibitory effects of adenosine (10-5 to 10-3 mol/L) on FCS-induced DNA synthesis in SMCs plated at a high density. Results (mean±SE) are expressed as percentage of control, defined as thymidine incorporation in the presence of 2.5% FCS (n=3 experiments, each in quadruplicate). Inset bar graph shows the same effects (mean±SE) of adenosine on thymidine incorporation in a representative experiment. *Significant difference (P<.01) from control (2.5% FCS); significant difference (P<.05) from adenosine without EHNA.

Modulatory Effects of EHNA on Adenosine- and Dipyridamole-Induced Inhibition of Growth
Treatment of growth-arrested SMCs (plated at high density) with exogenous adenosine inhibited thymidine incorporation (Fig 4, P>.01). Although adenosine at a concentration of 1 mmol/L completely inhibited DNA synthesis, it had only marginal or no inhibitory effects at 100 µmol/L (Fig 4). Since adenosine is rapidly metabolized by adenosine deaminase, it is likely that the reduced effects of adenosine are due to its rapid breakdown. Indeed, the inhibitory effects of adenosine became concentration dependent and were dramatically enhanced in the presence of the adenosine deaminase inhibitor EHNA (Fig 4, P<.01). In the presence of EHNA, adenosine inhibited FCS-induced thymidine incorporation by approximately 40% at concentrations as low as 10 µmol/L (Fig 4).

Similar to the effects of adenosine on DNA synthesis, treatment of SMCs for 4 days with adenosine inhibited FCS-induced cell number (Fig 5, P>.01). The inhibitory effects of adenosine on cell proliferation were significantly enhanced in the presence of EHNA (10 µmol/L, P<.05, Fig 5). The increase in cell number (percent of control) after 4 days of treatment with 2.5% FCS and in the presence of EHNA (10 µmol/L), adenosine (100 µmol/L), and adenosine (100 µmol/L) plus EHNA (10 µmol/L) was 100% for FCS, 78.8±4.87% for EHNA (P<.05 compared with 2.5% FCS), 65±0.88% for adenosine (P<.05 compared with 2.5% FCS), and 47.8±1.9% for adenosine plus EHNA (P<.05 compared with 2.5% FCS and FCS plus adenosine; Fig 5).

View larger version (56K): [in this window] [in a new window]   Figure 5. Effects of EHNA on the modulating effects of exogenous adenosine on 2.5% FCS-induced cell growth (cell number). EHNA (10 µmol/L) enhanced the inhibitory effects of adenosine (Ade, 100 µmol/L) on cell number. Results (mean±SE) are expressed as percentage of control, defined as cell number in SMCs treated with 2.5% FCS. Number of cells per well in controls (100%) after 4 days of treatment in separate experiments (mean±SE, n=3 experiments) was 2.8±0.2x104, 2.6±104, and 0.98x104. *Significant difference (P<.01) from control (2.5% FCS); significant difference (P<.05) from adenosine and from EHNA.

Compared with EHNA, an equimolar concentration of dipyridamole was more potent in inhibiting DNA synthesis (Fig 2, P<.05) and cell proliferation (Fig 3, P<.05). Since EHNA and dipyridamole induce adenosine levels by separate mechanisms, we studied whether their inhibitory effects are increased when SMCs are treated with dipyridamole in the presence of EHNA. The inhibitory effects of dipyridamole (0.1 µmol/L) on FCS-induced thymidine incorporation were significantly enhanced in the presence of EHNA (10 µmol/L, Fig 6, P<.05). Thymidine incorporation (percent of control) in SMCs treated with 2.5% FCS and in the presence of EHNA (10 µmol/L), dipyridamole (0.1 µmol/L), and dipyridamole (0.1 µmol/L) plus EHNA (10 µmol/L) was 100% for FCS, 82±2.3% for EHNA (P<.05 compared with 2.5% FCS), 75±0.7% for dipyridamole (P<.05 compared with 2.5% FCS), and 60±2% for dipyridamole plus EHNA (P<.05 compared with 2.5% FCS, FCS plus EHNA, and FCS plus dipyridamole; Fig 6).

View larger version (73K): [in this window] [in a new window]   Figure 6. Effects of EHNA on the modulating effects of dipyridamole on 2.5% FCS-induced thymidine incorporation (A) and cell growth (cell number) (B). A, EHNA (10 µmol/L) enhanced the inhibitory effects of dipyridamole (DIP, 0.1 mmol/L) on FCS-induced DNA synthesis. Results (mean±SE) are expressed as percentage of control, defined as thymidine incorporation in the presence of 2.5% FCS (n=3 experiments, each in quadruplicate). Thymidine incorporation (disintegrations per minute per well) in controls was (mean±SE, n=3-4) 4035±264, 8243±101, and 8410±153. B, EHNA (10 mmol/L) enhanced the inhibitory effects of dipyridamole (0.1 mmol/L) on cell number. Results (mean±SE) are expressed as percentage of control, defined as cell number in SMCs treated with 2.5% FCS. The number of cells in controls after 4 days of treatment with 2.5% FCS was (mean±SE, n=3) 2.7±0.2x104. *Significant difference (P<.01) from control (2.5% FCS); significant difference (P<.05) from EHNA and from adenosine.

Similar to the modulatory effects of EHNA on DNA synthesis, the inhibitory effects of dipyridamole (0.1 µmol/L) on FCS-induced cell proliferation were significantly enhanced in the presence of EHNA (10 µmol/L, P<.05, Fig 6). The increase in cell number (percent of control) after 4 days of treatment with 2.5% FCS in the presence of EHNA (10 µmol/L), dipyridamole (0.1 µmol/L), and EHNA (10 µmol/L) plus dipyridamole (0.1 µmol/L) was 100% for FCS, 78.8±4.87% for EHNA, 88.7±3.3% for dipyridamole, and 66±1% for EHNA plus dipyridamole (Fig 6).

Effects of EHNA and Dipyridamole on FCS-Induced DNA Synthesis in the Presence of A₂ Adenosine Receptor Antagonist (KF17837)
Using KF17837, which represents a prototype for a new class of selective A₂ receptor antagonists,⁴² we have recently shown that the inhibitory effects of adenosine on SMC growth are mediated via A₂ adenosine receptors.²³ To confirm that the effects of EHNA and dipyridamole on DNA synthesis were mediated via generation of endogenous adenosine and were not due to the compounds themselves, the effects of EHNA and dipyridamole on DNA synthesis were evaluated in the presence and absence of KF17837. The inhibitory effects of EHNA (10 µmol/L) and dipyridamole (0.1 µmol/L) on FCS-induced DNA synthesis were significantly reduced in the presence of KF17837 (1 nmol/L, Fig 7).

View larger version (57K): [in this window] [in a new window]   Figure 7. Effects of KF17837 (KF, 1 nmol/L) on the modulating effects of EHNA (10 µmol/L) and dipyridamole (DIP, 0.1 µmol/L) on 2.5% FCS-induced thymidine incorporation. KF17837 decreased the inhibitory effects of EHNA and dipyridamole on FCS-induced DNA synthesis. Results (mean±SE) are expressed as percentage of control, defined as thymidine incorporation in the presence of 2.5% FCS (n=3 experiments, each in quadruplicate) or 2.5% FCS plus KF17837 (1 nmol/L). Thymidine incorporation (disintegrations per minute per well) in controls was (mean±SE, n=3) 4208±65 and 3837±72, respectively. *Significant difference (P<.05) from control (2.5% FCS); significant difference (P<.05) from EHNA and from dipyridamole.

	Discussion

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Treatment of SMCs with EHNA elevated adenosine levels in culture medium. Application of both exogenous adenosine and adenosine generated endogenously by SMCs treated with EHNA or dipyridamole inhibited FCS-induced DNA synthesis and SMC proliferation. Furthermore, the inhibitory effects of EHNA and dipyridamole were significantly reduced in the presence of KF17837, an A₂ adenosine receptor antagonist. Thus, the present study demonstrates that cultured rat aortic SMCs synthesize adenosine and that SMC-derived adenosine inhibits FCS-induced growth of aortic SMCs in an autocrine/paracrine fashion.

Abnormal growth of vascular smooth muscle contributes to the structural changes associated with hypertension and atherosclerosis.^{1 2 3 4 5 6 7} Numerous studies have investigated the mechanism or mechanisms that contribute to these vaso-occlusive disorders, and the findings implicate a cascade of events involving autocrine/paracrine factors generated by blood cells and endothelium. The endothelium forms a barrier between blood cells and SMCs and normally influences vascular tone and SMC growth by simultaneously releasing contracting/growth-promoting factors (such as PDGF and angiotensin II) and relaxing/growth-inhibiting agents (such as nitric oxide). A balanced basal production of growth-promoting and growth-inhibiting factors maintains homeostasis within the vasculature. Endothelium removal results in smooth muscle proliferation,^{1 2 3 4 5 6 7} suggesting that an intact monolayer of confluent endothelium normally exerts a net inhibitory influence on the underlying smooth muscle.⁴³

Several vasoconstrictor substances are also growth factors,^{1 2 3 4 5 6 7} and vasodilators in general have growth-inhibitory effects on SMCs.^{6 7} For instance, it has been shown that nitric oxide, atrial natriuretic peptide, and prostaglandins inhibit SMC growth.^{3 5 7 11 12 13} However, much less attention has been focused on adenosine, which is also a potent vasodilator. Adenosine is synthesized in large amounts by endothelial cells,⁴⁴ and we have recently shown that SMCs can metabolize cAMP to generate adenosine via the cAMP-adenosine pathway.¹⁰ Moreover, adenosine induces several antivaso-occlusive effects by inhibiting platelet aggregation,¹⁵ neutrophil-endothelial interactions,¹⁶ norepinephrine release from nerve terminals, and renin release from juxtaglomerular cells,^{19 20 21 22} suggesting that adenosine may function as an endogenous antivaso-occlusive autacoid.

Although the application of exogenous adenosine inhibits PDGF- as well as FCS-induced growth of cultured SMCs,^{23 40} whether endogenous adenosine inhibits SMC growth has not been systematically investigated. The physiological effects of adenosine are governed in part by the rapid rate of elimination of adenosine from the extracellular space. Elimination of adenosine from the interstitial space is mediated by facilitated transport of adenosine into cells and also by the metabolism of adenosine to inosine by adenosine deaminase.^{34 35 36 37 38} Inhibition of the enzyme adenosine deaminase by EHNA, as well as inhibition of adenosine transport and metabolism by dipyridamole, has been shown to increase endogenous levels of adenosine.^{36 37 38} Hence, we used these two compounds in the present study to increase endogenous and/or extracellular levels of adenosine so as to evaluate the effects of endogenously generated adenosine on FCS-induced growth.

Our observation that FCS-induced DNA synthesis and cell proliferation in SMCs were significantly inhibited in the presence of EHNA suggests that endogenous adenosine inhibits SMC growth. This is further supported by our finding that adenosine was present in the media of cultured SMCs, and its levels were significantly induced in the presence of the adenosine deaminase inhibitor EHNA. To rule out direct effects of EHNA on SMC growth, we also investigated the effects of dipyridamole, a chemically dissimilar drug that increases endogenous adenosine levels.^{36 37 38} Compared with EHNA, dipyridamole was more potent in inhibiting FCS-induced SMC growth; however, in contrast to EHNA, no increase in extracellular adenosine was observed.

There are several explanations for the finding that no increase in exogenous adenosine was observed in the presence of dipyridamole. Dipyridamole is an inhibitor of cellular nucleoside transport; hence, it is possible that the transport of adenosine from the intracellular compartment to the medium is inhibited and endogenous adenosine inhibits SMC growth via an intracellular pathway. However, it is more likely that increased amounts of adenosine in response to dipyridamole were rapidly converted to inosine by adenosine deaminase, which is present in FCS. The latter notion is supported by our observation that compared with cells incubated in the presence of FCS, the basal extracellular levels of adenosine were significantly higher in cells incubated in the absence of serum (data not shown). Furthermore, when SMCs were treated with exogenous adenosine, only a fraction of adenosine, close to basal levels, was recovered in the medium. However, when adenosine was added to SMCs pretreated with EHNA or when EHNA was added to the cells alone, a dramatic increase in adenosine was observed in the medium. Since serum is prepared by clotting blood and red blood cells are well endowed with adenosine deaminase,^{34 35} it is possible that a substantial amount of adenosine deaminase is present in the serum and metabolizes adenosine to inosine. This is supported by our finding that adenosine levels were almost undetectable in samples in which adenosine (10 µmol/L) was incubated with 2.5% FCS (without cells) but were approximately 1500-fold higher in samples in which adenosine was incubated with FCS in the presence of EHNA or in the media without FCS (data not shown). Finally, the ability of dipyridamole to inhibit cell growth could be due to multiple effects. For instance, in addition to inhibiting adenosine uptake, dipyridamole has been shown to inhibit phosphodiesterase.^{36 37 38}

Our contention that the inhibitory effects of EHNA and dipyridamole on SMC growth are mediated via adenosine generation is further supported by our observation that the inhibitory effects of EHNA and dipyridamole on DNA synthesis were significantly reversed in the presence of KF17837, an A₂ adenosine receptor antagonist.^{29 42} Adenosine mediates its biological effects through at least four different receptors, ie, A₁, A_2a, A_2b, and A₃, and all four adenosine receptor subtypes belong to the G protein–coupled receptor superfamily.^{14 15 16 17 18 19 20 21 22 23 24 29 30 31 32 33} In vascular biology, A₁ and A₂ receptors appear to be the most important adenosine receptor subtypes, although participation of other receptor subtypes cannot be ruled out. We have recently shown that the inhibitory effects of exogenous adenosine are partially reversed by KF17837, which is an A₂ receptor antagonist.²³ Also, a selective A₁ agonist, N⁶-cyclopentyladenosine, inhibits cell growth only at high concentrations. A₂ receptors are positively coupled to adenylyl cyclase via G_s,²⁹ and intracellular cAMP inhibits SMC growth.⁴⁰ These findings, taken together with our observation that KF17837 partially reverses the inhibitory effects of EHNA and dipyridamole, strongly suggest that the inhibitory effects of adenosine are mediated in part via A₂ receptors.

How do our in vitro findings extrapolate to physiological situations in vivo, and can adenosine prevent SMC growth in vivo? Our finding that low concentrations of adenosine were able to inhibit SMC growth in the presence but not the absence of EHNA suggests that although adenosine effectively inhibits SMC growth, its effects are underestimated in the current series of experiments, as they were conducted in the presence of FCS. Under physiological conditions in vivo, most of the adenosine deaminase is localized within cells,^{34 35} and adenosine in the extracellular compartment will be available in active form to mediate the physiological inhibitory effects. Additionally, since adenosine is synthesized by smooth muscle cells as well as endothelial cells via multiple pathways, this would ensure substantial pharmacologically active steady-state levels of adenosine locally at the interface between endothelial and smooth muscle cells. In contrast, pathological conditions associated with decreased adenosine synthesis or increased adenosine deaminase leakage would reduce the pharmacologically active levels of adenosine, which would result in decreased antivaso-occlusive effects of adenosine. Indeed, preliminary data from our laboratory suggest that adenosine deaminase may participate in at least two disease states associated with increased risk of vaso-occlusive events, ie, sickle cell anemia and hypertension associated with aging (Dubey et al, 1995, unpublished data). However, future studies are needed to confirm or reject this role of adenosine deaminase.

In conclusion, we provide evidence that SMCs synthesize adenosine and that SMC-derived adenosine inhibits FCS-induced growth of SMCs in an autocrine/paracrine fashion. Our findings suggest that adenosine produced by vascular SMCs may play a vital role as a local antivaso-occlusive agent, and abnormal synthesis of adenosine by medial SMCs or increased catabolism of adenosine by adenosine deaminase may importantly contribute to the abnormal growth of SMCs observed in hypertension and atherosclerosis. Furthermore, agents that elevate endogenous adenosine could be clinically important in preventing the abnormal SMC growth and proliferation observed in hypertension and atherosclerosis, thus exerting beneficial effects on vascular structure.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium EHNA = erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride FCS = fetal calf serum PBS = phosphate-buffered saline PDGF = platelet-derived growth factor SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-40319 and HL-35909), Bethesda, Md.

	Footnotes

 
Previously published in abstract form (Hypertension. 1995;26:40).

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Bobik A, Campbell JH. Vascular derived growth factors: cell biology, pathophysiology and pharmacology. Pharmacol Rev. 1993;45:1-42. [Medline] [Order article via Infotrieve]

2. Jackson CL, Schwartz SM. Pharmacology of smooth muscle cell replication. Hypertension. 1992;20:713-736. [Abstract/Free Full Text]

3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]

4. Casscells W. Migration of smooth muscle and endothelial cells: critical events in restenosis. Circulation. 1992;86:723-729. [Free Full Text]

5. Luscher TF, Dubey RK. Endothelium and platelet-derived vasoactive substances: role in the regulation of vascular tone and growth. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. 2nd ed. New York, NY: Raven Press Publishers; 1995:609-629.

6. Gibbons GH. Autocrine-paracrine factors and vascular remodeling in hypertension. Curr Opin Nephrol Hypertens. 1993;2:291-298. [Medline] [Order article via Infotrieve]

7. Dzau VJ, Gibbons GH. Endothelium and growth factors in vascular remodeling of hypertension. Hypertension. 1991;18(suppl III):III-115-III-121.

8. Anfossi G, Cavalot F, Massucco P, Mattielo L, Mularoni E, Hahn A, Trovati M. Insulin influences immunoreactive endothelin release by human vascular smooth muscle cells. Metab Clin Exp. 1993;42:1081-1083.

9. Lindner V, Giachelli CM, Schwartz SM, Reidy MA. A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-B chain mRNA. Circ Res. 1995;76:951-957. [Abstract/Free Full Text]

10. Dubey RK, Mi Z, Jackson EK. Vascular smooth muscle cells metabolize cyclic-AMP to generate adenosine. Hypertension. 1995,26:576. Abstract.

11. Itoh H, Pratt RE, Dzau VJ. Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells. J Clin Invest. 1990;86:1690-1697.

12. Nilsson J, Olsson AG. Prostaglandin E inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor. Atherosclerosis. 1984;54:77-82.

13. Dubey RK, Jackson EK, Luscher TF. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell: role of cyclic nucleotides and angiotensin₁ receptors. J Clin Invest. 1995;96:141-149.

14. Headrick JP, Berne RM. Endothelium-dependent and -independent relaxations to adenosine in guinea pig aorta. Am J Physiol. 1990;259:H62-H67. [Abstract/Free Full Text]

15. Cristalli G, Vittori S, Thompson RD, Padgett WL, Shi D, Daly JW, Olsson RA. Inhibition of platelet aggregation by adenosine receptor agonists. Naunyn Schmiedebergs Arch Pharmacol. 1994;349:644-650. [Medline] [Order article via Infotrieve]

16. Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J Appl Physiol. 1994;76:5-13. [Abstract/Free Full Text]

17. Yen MH, Wu CC, Chiou WF. Partially endothelium-dependent vasodilator effect of adenosine in rat aorta. Hypertension. 1988;11:514-518. [Abstract/Free Full Text]

18. Vials A, Burnstock G. A2-purinoceptor-mediated relaxation in the guinea-pig coronary vasculature: a role for nitric oxide. Br J Pharmacol. 1993;109:424-429. [Medline] [Order article via Infotrieve]

19. De Mey J, Burnstock G, Vanhoutte PM. Modulation of the evoked release of noradrenaline in canine saphenous vein via presynaptic receptors for adenosine but not ATP. Eur J Pharmacol. 1979;55:401-405. [Medline] [Order article via Infotrieve]

20. Ekas RD Jr, Steenberg ML, Eikenburg DC, Lokhandwala MF. Presynaptic inhibition of sympathetic neurotransmission by adenosine in the rat kidney. Eur J Pharmacol. 1981;76:301-307. [Medline] [Order article via Infotrieve]

21. Jackson EK. Adenosine: a physiological brake on renin release. Annu Rev Pharmacol Toxicol. 1991;31:1-35. [Medline] [Order article via Infotrieve]

22. Murray RD, Churchill PC. Effects of adenosine receptor agonists in the isolated, perfused rat kidney. Am J Physiol. 1984;247:H343-H348. [Abstract/Free Full Text]

23. Dubey RK, Gillespie D, Osaka K, Suzuki F, Jackson EK. Adenosine inhibits growth of rat aortic smooth muscle cells: possible role of A_2b receptor. Hypertension. 1996;27:786-793. [Abstract/Free Full Text]

24. Jackson EK, Koehler M, Mi Z, Dubey RK, Tofovic SP, Carcillo JA, Jones GS. Possible role of adenosine deaminase in vasoocclusive diseases. J Hypertens. In press.

25. Schrader J. Formation and metabolism of adenosine and adenine nucleotides in cardiac tissue. In: Phillis JW, ed. Adenosine and Adenine Nucleotides as Regulators of Cellular Function. Boca Raton, Fla: CRC Press; 1991:55-69.

26. Gordon JL. Extracellular ATP: effects, sources and fate. Biochem J. 1986;233:309-319. [Medline] [Order article via Infotrieve]

27. Lloyd HG, Deussen A, Wuppermann H, Schrader J. The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart. Biochem J. 1988;252:489-494. [Medline] [Order article via Infotrieve]

28. Mi Z, Jackson EK. Metabolism of exogenous cyclic AMP to adenosine in the rat kidney. J Pharmacol Exp Ther. 1995;273:728-733. [Abstract/Free Full Text]

29. Dalziel HH, Westfall DP. Receptors for adenine nucleotides and nucleosides: sub classification, distribution, and molecular characterization. Pharmacol Rev. 1994;46:449-466. [Medline] [Order article via Infotrieve]

30. Norton ED, Jackson EK, Turner MB, Vinmani R, Forman MB. The effects of intravenous infusions of selective adenosine A1-receptor and A2-receptor agonists on myocardial reperfusion injury. Am Heart J. 1992;123:332-338. [Medline] [Order article via Infotrieve]

31. Thornton JD, Liu GS, Olsson RA, Downey JM. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation. 1992;85:659-665. [Abstract/Free Full Text]

32. Auchampach JA, Gross GJ. Adenosine A1 receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol. 1993;264:H1327-H1336. [Abstract/Free Full Text]

33. Yao Z, Gross GJ. Glibenclamide antagonizes adenosine A1 receptor-mediated cardioprotection in stunned myocardium. Circulation. 1993;88:235-244. [Abstract/Free Full Text]

34. Geiger JD, Padua RA, Nagy JI. Adenosine deaminase regulation of purine actions. In: Phillis JW, ed. Adenosine and Adenine Nucleotides as Regulators of Cellular Function. Boca Raton, Fla: CRC Press; 1991:71-84.

35. Fredholm BB, Sollevi A. The release of adenosine and inosine from canine subcutaneous adipose tissue by nerve stimulation and noradrenaline. J Physiol (Lond). 1981;313:351-367. [Abstract/Free Full Text]

36. German DC, Kredich NM, Bjornsson TD. Oral dipyridamole increases plasma adenosine levels in human beings. Clin Pharmacol Ther. 1989;45:80-84. [Medline] [Order article via Infotrieve]

37. Wang T, Mentzer RM, Van Wylen DGC. Interstitial adenosine with dipyridamole: effect of adenosine receptor blockade and adenosine deaminase. Am J Physiol. 1992;263:H552-H558. [Abstract/Free Full Text]

38. Conti JB, Blelardinelli L, Utterback DB, Curtis AB. Endogenous adenosine is an antiarrhythmic agent. Circulation. 1995;91:1761-1767. [Abstract/Free Full Text]

39. Mills I, Gewirtz H. Cultured vascular smooth muscle cells from porcine coronary artery possess A1 and A2 adenosine receptor activity. Biochem Biophys Res Commun. 1990;168:1297-1302. [Medline] [Order article via Infotrieve]

40. Jonzon B, Nilsson J, Fredholm BB. Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells. J Cell Physiol. 1985;124:451-456. [Medline] [Order article via Infotrieve]

41. Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth muscle cells: mitogenic responses to angiotensin II. Circ Res. 1992;71:1143-1152. [Abstract/Free Full Text]

42. Shimada J, Suzuki F, Nonaka H, Ishii A, Ichikawa S. (E)-1,3-Dialkyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanthines: potent and selective adenosine A₂ antagonists. J Med Chem. 1992;35:2342-2345. [Medline] [Order article via Infotrieve]

43. DeMey JGR, Dijkstra EH, Vrijdag MJJF. Endothelium reduces DNA synthesis in isolated arteries. Am J Physiol. 1991;260:H1128-H1134. [Abstract/Free Full Text]

44. Smolenski RT, Kochan ZK, McDouall R, Page C, Seymour A-ML, Yacoub MH. Endothelial nucleotide catabolism and adenosine production. Cardiovasc Res. 1994;28:100-104.[Abstract/Free Full Text]

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