Exogenous and Endogenous Adenosine Inhibits Fetal Calf Serum–Induced Growth of Rat Cardiac Fibroblasts
Role of A2B Receptors
Background Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast–derived) adenosine on cardiac fibroblast proliferation.
Methods and Results Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues.
Conclusions Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast–derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast–derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.
Cardiac fibroblasts are importantly involved in the pathophysiology of cardiac remodeling induced by hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.1 Cardiac fibroblasts contribute to pathological structural changes in the heart by undergoing proliferation, depositing extracellular matrix proteins, and replacing myocytes with fibrotic scar tissue.1 Thus, cardiac fibroblast–induced cardiac remodeling may participate in diastolic and systolic dysfunction, leading to congestive heart failure.
A number of autocrine/paracrine factors1 as well as physical forces such as mechanical stretch2 can stimulate cardiac fibroblast growth. In a normal heart, however, quiescence is maintained by a balance between circulating and cardiac-derived inhibitors and promoters of cardiac fibroblast growth.3 Disruption of the balanced generation of growth promoters and inhibitors under pathological conditions could trigger an increased proliferation of cardiac fibroblasts, enhanced deposition of extracellular matrix by cardiac fibroblasts, and enlargement and stiffening of the heart. Therefore, autocrine/paracrine factors generated by cells within the heart wall that inhibit cardiac fibroblast growth may play a major cardioprotective role.
In this regard, adenosine may be an important antiproliferative factor. Adenosine is synthesized by the cardiac wall and exerts numerous cardioprotective4 and anti–vaso-occlusive5 actions. Cardiomyocytes,6 vascular SMCs,7 8 and endothelial cells,9 10 both vascular9 11 and cardiac,9 10 have several metabolic pathways for generating large amounts of adenosine. For example, it has been shown that endothelial cells synthesize adenosine and have an adenine pool that is two to three times greater than that of hepatocytes.10 11 Moreover, we have recently shown that cardiac fibroblasts, which constitute 60% of the total heart cells,12 can also synthesize adenosine from exogenous cAMP.13
Adenosine has long been known as a “retaliatory” metabolite, particularly in the heart, where it induces cardioprotective effects.13 Furthermore, adenosine has several anti–vaso-occlusive properties; for instance, adenosine induces vasodilation,14 inhibits platelet aggregation,15 prevents neutrophil adhesion to vascular and cardiac endothelial cells,16 attenuates neutrophil-induced endothelial cell damage,16 stimulates nitric oxide release from vascular endothelial cells,17 activates the cellular antioxidant defense system18 and prevents oxygen free radical–induced injury,16 and blocks the synthesis of potent mitogenic factors such as angiotensin II and norepinephrine by inhibiting renin release19 and noradrenergic neurotransmission.20 Moreover, we have recently shown that both exogenous and SMC-derived adenosine inhibits FCS-induced growth of SMCs.21 Because several factors regulating SMC growth have similar effects on cardiac fibroblast growth, we hypothesize that adenosine may be a potential endogenous factor that is important in regulating cardiac fibroblast growth and maintaining cardiac homeostasis.
Accordingly, the aims of the present study were (1) to determine whether exogenous adenosine inhibits growth of cardiac fibroblasts, (2) to identify the adenosine receptor subtype(s) involved in mediating any antiproliferative effects of adenosine on cardiac fibroblasts, (3) to determine whether cardiac fibroblasts synthesize adenosine, and (4) to determine whether cardiac fibroblast–derived adenosine can effectively inhibit cardiac fibroblast growth. To accomplish these aims, it was necessary to stimulate cardiac fibroblast growth, for which purpose FCS was selected. The rationale for choosing FCS was that it is the most potent growth stimulator, containing a battery of growth factors, such as platelet-derived growth factor, fibroblast growth factor, angiotensin II, endothelin, and norepinephrine, that have been implicated in the pathophysiology of cardiac remodeling in hypertension and myocardial infarction. To investigate the synthesis and antiproliferative properties of endogenous adenosine, it was necessary to prevent the metabolism of cardiac fibroblast–derived adenosine. In the present study, this was accomplished by inhibiting (1) adenosine deaminase with EHNA, (2) adenosine kinase with IDO, and (3) adenosine uptake with DIP.
DMEM, DMEM/F12 medium, HBSS, penicillin, streptomycin, 0.25% trypsin-EDTA solution, collagenase, and all tissue culture ware were purchased from GIBCO Laboratories. FCS was obtained from HyClone Laboratories Inc. Adenosine, Cl-Ad, EHNA, and DIP were purchased from Sigma Chemical Co. CPA, CGS21680, DPCPX, IDO, DPSPX, NECA, and MECA were purchased from Research Biochemicals International. KF17837 was a generous gift from Dr F. Suzuki, Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co Ltd, Sunto, Shizuoka, Japan. [3H]Thymidine (specific activity, 11.8 Ci/mmol) was purchased from ICN Biomedicals. All other chemicals used were of tissue culture grade or best grade available.
Cardiac Fibroblast Cell Culture
Sprague-Dawley male rats (n=12) weighing 150 to 200 g were obtained from Charles River (Wilmington, Mass) and were fed standard rat chow and tap water ad libitum. Both left ventricular and atrial cardiac fibroblasts were cultured by the method of Farivar et al22 using the enzymatic digestion and selective plating technique, with minor modifications. Briefly, hearts were obtained from ether-anesthetized rats via a midline abdominal incision, including the diaphragm. The atria and left ventricle were removed and minced separately, and the minced sections were washed twice with DMEM. The sections were suspended in 5 mL of collagenase type II in DMEM and incubated at 37°C for 1 hour in a shaking water bath. The supernatant obtained after 1 hour of incubation was discarded, and the tissue was incubated further in fresh collagenase solution for 1 hour. After collagenase digestion, the dissociated cells were centrifuged, and the pellet was suspended in complete culture medium (DMEM/F12 supplemented with HEPES 25 mmol/L). Cells were plated in tissue culture flasks (75 cm2) and incubated under standard tissue culture conditions (37°C, 5% CO2/95% air, and 98% humidity). Because cardiac fibroblasts adhere to culture surfaces faster than myocytes and endothelial cells, cardiac fibroblasts could be separated from endothelial cells and myocytes by selective plating. Briefly, 2 hours after plating, the nonadherent cells in the supernatant were removed after gentle shaking, and the flasks were washed once with complete culture medium. The attached cardiac fibroblasts were subsequently fed complete culture medium and allowed to grow under standard tissue culture conditions. The cardiac fibroblasts grew robustly and were confluent in 5 to 7 days. Confluent monolayers of cardiac fibroblasts were dislodged by treatment with 0.25% trypsin-EDTA solution and passaged further. Cardiac fibroblasts in the second and third passages were used in all studies.
Purity of the cardiac fibroblasts was assessed by immunostaining and by examination of the morphology of the cells at the second and third passages. More than 98% of the cells had fibroblast morphology, ie, were thin and triangular with a light cytoplasm. Myocytes, which are large, contain birefringent nuclei and dark cytoplasm-containing granules and myofibrils, and contract rhythmically in culture, and endothelial cells, which have a characteristic cobblestone morphology, were absent in the cultures. Negative staining was observed in cells treated with polyclonal antibody against von Willebrand factor (factor VIII) and with monoclonal antibodies against sarcomeric (striated muscle) actin and desmin, suggesting the absence of endothelial cells, SMCs, and myocytes. Moreover, reaction with polyclonal anti-vimentin antibody positively stained >99% of monolayers. Taken together, these findings suggest that the cultured cells were pure cardiac fibroblasts.
[3H]Thymidine incorporation studies were done to investigate the effects of agents on FCS-induced DNA synthesis. Atrial or ventricular cardiac fibroblasts were plated at a density of 2.5×104 cells/well in 24-well tissue culture dishes and allowed to grow for 48 hours in complete culture medium containing 10% FCS under standard tissue culture conditions. The cells were then growth-arrested by feeding with complete culture medium containing 0.4% BSA (Sigma) for 48 hours. Growth was initiated by treatment of growth-arrested cells for 20 hours with complete culture medium supplemented with 2.5% FCS and containing or lacking adenosine receptor agonists or antagonists or modulators of adenosine metabolism (Table⇓) as follows: adenosine, Cl-Ad, CPA, CGS21680, NECA, MECA, Cl-Ad plus DPCPX, Cl-Ad plus KF17837, Cl-Ad plus DPSPX, EHNA, DIP, IDO, EHNA plus IDO, DIP plus IDO, DIP plus IDO plus EHNA, KF17837, EHNA plus KF17837, DIP plus KF17837, DPSPX, EHNA plus DPSPX, DIP plus DPSPX, DPCPX, EHNA plus DPCPX, or DIP plus DPSPX. After 20 hours of incubation, 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 two washings of the cells with Dulbecco’s PBS and two with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 μL of 0.3N NaOH and 0.1% SDS after incubation at 50°C for 2 hours. Aliquots from 4 wells for each treatment with 10 mL scintillation fluid were counted in a liquid scintillation counter. Each experiment was repeated four to six times.
Cell Proliferation (Cell Number)
Trypsinized cardiac fibroblasts 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 1×104 cells/well. After incubation for 18 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 adenosine on FCS-induced cytokinesis, we treated growth-arrested cardiac fibroblasts every 24 hours for 4 days with complete culture medium containing 2.5% FCS and supplemented with or lacking adenosine, Cl-Ad, CPA, CGS, NECA, MECA, EHNA, DIP, IDO, EHNA plus DIP, EHNA plus IDO, DIP plus IDO, or DIP plus IDO plus EHNA. The treatments were terminated on day 5, and cells were dislodged with trypsin-EDTA, diluted in Isoton-II, and counted with a hemocytometer-calibrated Coulter counter. Aliquots from 4 wells were counted for each group, and four to six independent experiments were performed for each treatment.
Adenosine Synthesis by Cardiac Fibroblasts: Effects of EHNA, IDO, and DIP
To evaluate whether atrial and ventricular cardiac fibroblasts synthesize adenosine, we assayed adenosine synthesis by cultured rat cardiac fibroblasts in the presence and absence of EHNA, IDO, and DIP to augment endogenous adenosine levels. Briefly, cardiac fibroblasts (atrial or ventricular, third passage) were plated in 12-well culture plates and grown to confluency by feeding with complete culture medium containing 10% FCS. On the day of the experiment, confluent monolayers of cells were washed twice with Dulbecco’s PBS and then incubated with buffered PBS (Dulbecco’s PBS, HEPES 25 mmol/L, NaHCO3 13 mmol/L) supplemented with 2.5% FCS and containing or lacking EHNA, DIP, IDO, EHNA plus DIP, EHNA plus IDO, DIP plus IDO, or DIP plus IDO plus EHNA. After 4 hours of incubation, the supernatants were withdrawn and transferred into ice-cold microcentrifuge tubes and frozen at −70°C until adenosine levels were estimated. After the collection of supernatants, the monolayers of cells were inspected microscopically for intactness, and the numbers of cells were counted.
To evaluate whether cardiac fibroblasts catabolize adenosine, we also assayed the levels of adenosine in the medium of confluent monolayers of cardiac fibroblasts treated for 4 hours with exogenous adenosine (10 μmol/L) in the presence and absence of EHNA, IDO, or EHNA plus IDO. All experiments were conducted in nine separate cultures. The data were normalized to cell numbers, which were counted after the medium was collected.
Adenosine levels in the samples were analyzed by HPLC via our previously described method.23 Briefly, samples were thawed and centrifuged at 10 000 rpm for 5 minutes. Supernatant (80 μL) was injected into an Isco HPLC system (pump model 2350, gradient programmer model 2360, V4 absorbance detector, 4.6×250-mm C18 column with 5-μm particle size, and ChemResearch Data Management System). Mobile phase A was KH2PO4 (0.1 mol/L, pH 6.1) and mobile phase B was 80% KH2PO4 (0.01 mol/L, 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 quantified as the area under the chromatographic peak, and the absolute amount in each sample was calculated from a standard curve of adenosine.
All growth experiments were performed in triplicate or quadruplicate with four to six separate cultures, whereas adenosine synthesis and catabolism experiments were performed with seven to nine separate cultures. Data for the DNA synthesis, cell number, and adenosine levels in the medium are presented as mean±SEM. Statistical analysis was performed with ANOVA, paired Student’s t test, or Fisher’s least significant difference test as appropriate. A value of P<.05 was considered statistically significant.
Effect of Exogenous Adenosine and Cl-Ad on FCS-Induced Growth of Atrial Cardiac Fibroblasts
Compared with the growth-arrested cells, treatment with 2.5% FCS stimulated DNA synthesis by 7- to 10-fold (P<.001; data not shown). Both adenosine and Cl-Ad inhibited FCS-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig 1⇓). Compared with adenosine, Cl-Ad was more potent in inhibiting FCS-induced thymidine incorporation. The lowest concentrations of adenosine and Cl-Ad that significantly inhibited FCS-induced DNA synthesis were 10 and 0.1 nmol/L, respectively. A 50% decrease in FCS-induced thymidine incorporation by adenosine and Cl-Ad was observed at 400 and 6 μmol/L, respectively (Fig 1⇓).
The density of the cells during exposure to an agent can alter its response because the rate of catabolism would depend on the number of cells present. Therefore, we compared the effects of adenosine on FCS-induced thymidine incorporation in cells plated at a high density (2.5×104 cells/well) versus a lower density (5×103 cells/well). Additionally, to test whether catabolism of adenosine by adenosine deaminase21 is responsible for altering/reducing the inhibitory effects of adenosine, we studied the effects of adenosine on FCS-induced DNA synthesis in cells in the presence and absence of EHNA.
The inhibitory effects of adenosine on FCS-induced DNA synthesis were significantly increased when cells plated at the lower density were treated with adenosine (Fig 2⇓). Low concentrations of adenosine (0.1 nmol/L) inhibited FCS-induced thymidine incorporation by ≈20% in cells plated at low density but not in cells plated at high density. Moreover, the inhibitory effects of adenosine were significantly enhanced in the presence of EHNA (Fig 2⇓). To evaluate whether adenosine in the cells and/or medium is metabolized by adenosine deaminase and/or adenosine kinase, the levels of adenosine in the medium of cells treated with adenosine 10 μmol/L in the presence and absence of EHNA, IDO, or EHNA plus IDO were measured. As shown in Fig 3⇓, the levels of adenosine in the medium of cells treated with adenosine alone were close to those observed in controls (cells treated with vehicle). However, in cells treated with adenosine in the presence of the adenosine deaminase inhibitor EHNA or the adenosine kinase inhibitor IDO, a significant increase in the recovery of adenosine in the medium was observed (Fig 3⇓). Moreover, in cells treated with EHNA plus IDO, a dramatic increase in the recovery of adenosine in the medium was observed (2511-fold higher than cells treated with adenosine alone; Fig 3⇓).
FCS induced proliferation (cell number) of growth-arrested atrial cardiac fibroblasts by 9- to 11-fold. Adenosine and Cl-Ad inhibited FCS-induced increase in cell number in a concentration-dependent manner (Fig 1⇑). Like the effects on DNA synthesis, Cl-Ad was more potent in inhibiting cell proliferation than was adenosine. Trypan blue exclusion tests indicated no loss in viability of cells treated with adenosine. In cells treated with Cl-Ad, cell toxicity was observed at the maximal concentration of Cl-Ad used (10−3 mol/L); however, at lower concentrations (10−10 to 10−4 mol/L), no loss in cell viability was evident (data not shown).
Effect of Atrial Cardiac Fibroblast–Derived Adenosine on FCS-Induced Cell Growth
To evaluate the effects of endogenous cardiac fibroblast–derived adenosine on cell growth, we assayed the effects of EHNA, IDO, and DIP on FCS-induced cardiac fibroblast growth as well as on adenosine synthesis. Adenosine levels were nondetectable in samples drawn at time zero; however, the levels of adenosine were significantly increased in the medium of cardiac fibroblasts collected after 4 hours of incubation (Fig 3⇑). Compared with untreated controls, treatment of cells with EHNA, DIP, or IDO increased the levels of adenosine in the medium (Fig 3⇑). Moreover, in cells treated with EHNA+IDO or EHNA+IDO+DIP, the levels of adenosine were increased >100-fold compared with cells treated only with EHNA or IDO (Fig 3⇑).
EHNA, DIP, and IDO inhibited FCS-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig 4⇓). The lowest concentrations of EHNA, DIP, and IDO that significantly inhibited FCS-induced DNA synthesis were 10, 0.01, and 0.01 μmol/L, respectively. A 50% decrease in FCS-induced thymidine incorporation by EHNA, DIP, and IDO was observed at ≈50, 3, and 1 μmol/L, respectively (Fig 4⇓).
Like the effects on DNA synthesis, EHNA, DIP, and IDO inhibited FCS-induced increase in cell number in a concentration-dependent manner and in the following order of potency: DIP=IDO>EHNA (Fig 5⇓). The lowest concentrations of DIP, IDO, and EHNA that inhibited cell proliferation were 0.01, 0.01, and 10 μmol/L, respectively (Fig 5⇓). Half-maximal effects of EHNA, DIP, and IDO on cell proliferation were observed at 10 to 25, 0.1, and 0.1 μmol/L, respectively (Fig 5⇓). Because EHNA, IDO, and DIP induce adenosine levels by separate mechanisms, we studied their combined inhibitory effects on cell growth. As shown in Fig 6⇓, the inhibitory effects of EHNA, DIP, and IDO on thymidine incorporation and cell number were greater with combinations of these agents than with the individual agents. Moreover, in cells treated with EHNA plus DIP plus IDO, FCS-induced cell proliferation was completely blocked.
Effect of Receptor-Selective Adenosine Analogues (CPA, CGS21680, NECA, and MECA) on FCS-Induced Growth of Atrial Cardiac Fibroblasts
High (10−4 mol/L) but not low concentrations of CPA and CGS21680 inhibited FCS-induced DNA synthesis and cell proliferation (Fig 7⇓). NECA was more potent than CPA and CGS21680 but less potent than MECA. Although NECA was more potent than CGS21680 and CPA in inhibiting thymidine incorporation and cell proliferation, it was significantly less potent than Cl-Ad. In contrast, MECA was as potent as Cl-Ad in inhibiting FCS-induced increases in cell number (compare Fig 1⇑ with Fig 7⇓).
Effects of A1 (DPCPX) and A2 Adenosine Receptor Antagonists (DPSPX, KF17837) on Cl-Ad–, EHNA-, and DIP-Induced Inhibition of DNA Synthesis in Atrial Cardiac Fibroblasts
Because CGS21680 had little effect on FCS-induced growth and CPA was less potent than NECA, MECA, and Cl-Ad, the possible involvement of A1 and A2A receptor in mediating the inhibitory effects of adenosine could be ruled out (Table⇑). Further experiments were conducted using the adenosine receptor antagonists DPCPX, DPSPX, and KF17837, which inhibit the effects of adenosine by blocking A1, A1+A2, and A2 receptors, respectively (Table⇑). KF17837 and DPSPX but not DPCPX significantly reversed the inhibitory effects of Cl-Ad on FCS-induced DNA synthesis (Fig 8⇓).
We also determined the effects of EHNA and DIP on DNA synthesis in the presence and absence of adenosine receptor antagonists (DPCPX, DPSPX, and KF17837). The inhibitory effects of EHNA and DIP on FCS-induced DNA synthesis were significantly attenuated by the A2 receptor antagonists KF17837 and DPSPX (Fig 9⇓) but not by the A1 receptor antagonist DPCPX (Fig 8⇑). These data confirm that the inhibitory effects of EHNA and DIP were mediated via generation of endogenous adenosine and that the inhibitory effects of atrial cardiac fibroblast–derived adenosine on cell growth were mediated via A2 adenosine receptors. Trypan blue exclusion tests indicated no loss in viability of cells treated with CPA, CGS21680, MECA, NECA, DPSPX, KF17837, or DPCPX, and <0.5% took up the dye (data not shown).
Effects of Exogenous and Endogenous Adenosine on FCS-Induced DNA Synthesis in Cardiac Fibroblasts Cultured From Left Ventricles
Because the abnormal growth of cardiac fibroblasts in the left ventricle also contributes to cardiac remodeling, we investigated whether exogenous and endogenous adenosine inhibits FCS-induced DNA synthesis in left ventricular cardiac fibroblasts. Adenosine and its analogues with A2B receptor activity, including Cl-Ad, MECA, and NECA, but not agonists with A1 (CPA) or A2A (CGS21680) receptor activity inhibited FCS-induced thymidine incorporation (Fig 10A⇓). Adenosine analogues inhibited ventricular cardiac fibroblasts in the following order of potency:
Cl-Ad=MECA >adenosine>NECA >CPA >CGS21680 (Fig 10A⇑).
As in atrial cardiac fibroblasts, treatment with EHNA, DIP, and IDO increased exogenous adenosine levels in left ventricular cardiac fibroblasts (Fig 10B⇑) and inhibited FCS-induced DNA synthesis (Fig 10C⇑). The combined effects of EHNA, IDO, and DIP on adenosine levels (Fig 10B⇑) and FCS-induced DNA synthesis (Fig 10C⇑) in ventricular cardiac fibroblasts were greater than the effects of these agents alone. To confirm that the inhibitory effects of EHNA and DIP are due to increases in adenosine and that adenosine mediates its effects via A2 receptors, we studied the effects of Cl-Ad, EHNA, and DIP on DNA synthesis in the presence and absence of DPSPX, KF17837, and DPCPX. The inhibitory effects of Cl-Ad, EHNA, and DIP were reversed by DPSPX and KF17837 but not by DPCPX (Fig 11⇓).
To rule out the possibility that serum-derived adenosine contributes to the inhibitory effects, we assayed adenosine levels in serum (2.5% in PBS) treated or not treated with EHNA, DIP, IDO, or EHNA+IDO in the presence and absence of exogenous adenosine (Fig 12⇓). Adenosine levels were below detection limit in serum and serum plus EHNA, DIP, IDO, or EHNA+IDO. Moreover, adenosine levels were almost undetectable in samples in which exogenous adenosine (10 μmol/L) was incubated with 2.5% FCS but were ≈6000- to 6600-fold higher in samples in which adenosine was incubated with FCS in the presence of EHNA or in the media without FCS. Compared with EHNA, IDO and DIP were less effective in preventing FCS-induced breakdown of adenosine. These results suggest that a substantial amount of adenosine deaminase is present in the serum and is responsible for catabolizing adenosine to inosine. This is further supported by the fact that serum is prepared by clotting blood, and red blood cells contain significant amounts of adenosine deaminase.
The results of the present study demonstrate that adenosine inhibits FCS-induced growth of atrial and left ventricular cardiac fibroblasts. Treatment of cardiac fibroblasts with adenosine, with a stable adenosine analogue (Cl-Ad), and with agents that elevate endogenous adenosine (EHNA, IDO, DIP) inhibited FCS-induced DNA synthesis and cell proliferation. The potency of MECA, an adenosine agonist with high affinity for A2 receptors,24 was similar to that of Cl-Ad, and the potency of NECA, an adenosine agonist with equal affinity for both A1 and A2 receptors,25 was less than that of MECA. The adenosine agonists CPA and CGS21680, which are selective A1 and A2A receptor agonists,25 respectively, were only weakly inhibitory. Thus, the inhibitory effects of adenosine are most likely mediated via A2B receptors and not via A1 or A2A receptors. Furthermore, the inhibitory effects of Cl-Ad, EHNA, and DIP were significantly reversed by KF17837, a selective A2 receptor antagonist,26 and by DPSPX, a nonselective A2 receptor antagonist,27 but not by DPCPX, an A1 receptor antagonist.25 Taken together, our findings provide the first evidence that exogenous as well as cardiac fibroblast–derived adenosine inhibits serum-induced growth in an autocrine/paracrine fashion and via the A2B receptor.
The fact that cardiac fibroblast quiescence is maintained in a normal heart suggests that the growth-inhibitory effects dominate and are important for maintaining homeostasis in the heart. Therefore, it is feasible that decreased production of growth inhibitors (such as adenosine) may tilt the balance toward cardiac fibroblast growth, which would result in increased growth of cardiac fibroblasts, leading to cardiac remodeling, enlargement of the heart, and cardiac dysfunction. Hence, detailed knowledge of the roles of different endogenous inhibitors that regulate cardiac fibroblast growth and maintain homeostasis is of great clinical and therapeutic importance. In this regard, the role of the vasodilator adenosine has not been well investigated.
Structural changes in the heart probably involve multiple autocrine/paracrine/endocrine factors.1 2 3 FCS contains a battery of growth factors, including platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, angiotensin II, endothelin, and norepinephrine, that may contribute to the cardiac remodeling process. Therefore, we thought it important to evaluate the effects of adenosine on FCS-induced growth of cardiac fibroblasts so as to elucidate the growth-regulatory effects of adenosine under more physiological conditions. The finding that adenosine inhibits FCS-induced cardiac fibroblast growth provides the first evidence that adenosine is an important modulator of cardiac fibroblast growth.
Our observation that CPA (an adenosine analogue that is highly selective for A1 receptors25 and mediates its effects at pharmacologically low doses [<10−9 mol/L]) was unable to inhibit FCS-induced growth of cardiac fibroblasts at low concentrations suggests that the inhibitory effects of adenosine are not mediated via A1 receptors. This conclusion is further supported by our observation that DPCPX, an adenosine receptor antagonist that is 700-fold selective for A1 receptors,25 was unable to block the inhibitory effects of Cl-Ad on FCS-induced growth of cardiac fibroblasts. The observation that high concentrations of CPA inhibited FCS-induced growth of cardiac fibroblasts suggests the possible involvement of some receptor with low affinity for CPA. It has been shown that although CPA at low concentrations mediates its effects selectively via A1 receptors, high concentrations of CPA can activate both A2 and A4 adenosine receptors.25 Hence, it is possible that the inhibitory effects of CPA on cardiac fibroblast growth at high concentrations are mediated in part via either A2 or A4 adenosine receptors.
The finding that KF17837 and DPSPX but not DPCPX effectively reversed the inhibitory effects of Cl-Ad suggests that the inhibitory effects of adenosine are A2 receptor mediated. Moreover, our observations that (1) CGS21680 is ineffective in mimicking the inhibitory effects of adenosine, (2) MECA was as effective as Cl-Ad in inhibiting cardiac fibroblast growth, and (3) NECA was more effective than CGS21680 but less effective than Cl-Ad and MECA provide strong evidence that the effects of adenosine are not mediated via A2A receptors. Rather, our data suggest that the A2B receptors may be involved in mediating the inhibitory effects of adenosine.
Our contention that the inhibitory effects of adenosine are mediated via A2B receptors is further supported by the recently proposed and endorsed subclassification of A2A and A2B receptors.25 Gurden et al28 demonstrated that the relative potencies of CGS21680 and NECA can be used as a reference to differentiate A2A from either A2B or A1 receptors. When the effects of CGS21680 are as potent as NECA, this implicates the A2A receptor. However, when CGS21680 is much less potent than NECA, this indicates that the observed effects are mediated via activation of the A2B receptor subtype. In the present study, compared with CGS21680, NECA was more effective in mimicking the inhibitory effects of adenosine, which further substantiates our conclusion that the inhibitory effects of adenosine are mediated via the A2B receptor.
A2 receptors are positively coupled with adenylyl cyclase, and their activation results in a significant increase in cAMP levels.25 Stimulation of cardiac fibroblasts with adenosine has been shown to elevate cAMP levels, and cAMP in turn has antiproliferative effects on cardiac fibroblasts.29 Therefore, the inhibitory effects of adenosine on cardiac fibroblast growth are most likely mediated largely via the second messenger cAMP; however, the participation of other mechanism(s) cannot be ruled out. Activation of A2B receptors by adenosine stimulates NO release from endothelial cells,17 and we have recently observed that adenosine amplifies lipopolysaccharide-induced NO release from vascular SMCs.30 Because NO inhibits cardiac fibroblast proliferation,3 this provides an additional pathway through which adenosine could inhibit cardiac fibroblast growth.
The above findings provide the first evidence that exogenous adenosine inhibits FCS-induced growth of cardiac fibroblasts and that the inhibitory effects of adenosine are mediated via activation of A2B receptors. However, whether endogenous adenosine also inhibits cardiac fibroblast growth cannot be inferred from studies with agonists. Therefore, we examined the growth-inhibiting effects of agents that elevate cellular adenosine levels via different mechanisms to assess the role of endogenous, ie, cardiac fibroblast–derived, adenosine on cardiac fibroblast growth.
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, by the metabolism of adenosine to inosine by adenosine deaminase,31 32 and by the metabolism of adenosine to AMP by adenosine kinase.32 Inhibition of the enzyme adenosine deaminase by EHNA and the enzyme adenosine kinase by IDO as well as the inhibition of adenosine transport and metabolism by DIP has been shown to increase endogenous levels of adenosine.31 Hence, these three compounds were used in the present study to increase endogenous levels of adenosine so as to evaluate the effects of endogenously generated adenosine on FCS-induced growth.
Treatment of cardiac fibroblasts with EHNA, IDO, and DIP elevated the levels of adenosine in the culture medium and inhibited FCS-induced DNA synthesis and proliferation of cardiac fibroblasts. Thus, the present study demonstrates that cultured rat cardiac fibroblasts synthesize adenosine and that cardiac fibroblast–derived adenosine inhibits FCS-induced growth of cardiac fibroblasts in an autocrine fashion.
The observation that adenosine was present in the media of cultured cardiac fibroblasts provides evidence for basal synthesis of adenosine in these cells. This is further supported by the fact that adenosine levels increased dramatically in the presence of EHNA and IDO. We have previously demonstrated that cardiac fibroblasts can generate adenosine by metabolizing cAMP via the cAMP-adenosine pathway.29 However, in addition to the cAMP-adenosine pathway, three other distinct pathways for adenosine biosynthesis have been well established. (1) The intracellular ATP pathway involves sequential dephosphorylation of ATP to adenosine (ATP→ADP→AMP→adenosine) within the cell and is dependent on the balance between energy supply and demand.33 (2) The extracellular ATP pathway, which is not dependent on an imbalance between energy supply and demand, is a source of extracellular adenosine regardless of the metabolic demands of the cell.33 In this regard, release of adenine nucleotides (ATP, ADP, AMP) from sympathetic nerve terminals, adrenal chromaffin cells, platelets, and endothelial cells provides an extracellular ATP pathway in which ectoenzymes (ecto-ATPases, ecto-ADPases, and ecto-5′-nucleotidase) convert ATP to adenosine.33 (3) The transmethylation pathway, which involves the hydrolysis of S-adenosyl-l-homocysteine to l-homocysteine and adenosine, is also involved.33 Unlike the intracellular and extracellular ATP pathways, the transmethylation reaction is relatively constant and provides a basal tone of adenosine production that does not depend on crisis events, such as ischemia, platelet activation, and neutrophil attack. The biosynthetic pathways involved in the basal synthesis of adenosine by cardiac fibroblasts cannot be deduced from the present study.
In cardiac fibroblasts treated with EHNA plus IDO, the adenosine levels increased by >690-fold. This suggests that both adenosine deaminase and adenosine kinase regulate adenosine elimination in cardiac fibroblasts. Direct evidence for this notion comes from our observation that when cardiac fibroblasts were treated with exogenous adenosine, only a fraction of adenosine, close to basal levels, was recovered in the medium. However, in the presence of EHNA plus IDO but not in the presence of EHNA or IDO alone, a dramatic increase in the recovery of adenosine was observed.
Our contention that the inhibitory effects of the modulators of endogenous adenosine on cardiac fibroblast growth are mediated via generation of adenosine is supported by our observation that the inhibitory effects of EHNA and DIP on DNA synthesis were significantly reversed by KF17837 and DPSPX. Moreover, the inhibitory effects of EHNA, IDO, and DIP were significantly enhanced when cardiac fibroblasts were treated with a combination of these agents. DPCPX did not reverse the inhibitory effects of EHNA and DIP on cardiac fibroblasts, which strongly suggests that the inhibitory effects of endogenous adenosine are mediated via A2 receptors.
As shown in Figs 3⇑ and 10⇑, IDO, EHNA, and DIP at 1, 10, and 0.1 μmol/L, respectively, caused similar increases in adenosine, and as shown in Figs 4⇑ and 10⇑, these same concentrations cause similar inhibition of DNA synthesis. However, as shown in Fig 5⇑, these concentrations of IDO, EHNA, and DIP had somewhat disparate effects on cell number. There are several explanations for the finding that DIP and IDO were somewhat more effective than EHNA in inhibiting cardiac fibroblast proliferation even though the observed increase in adenosine levels in the medium were similar. Most likely, measurements of extracellular adenosine in the bulk medium in response to DIP and IDO underestimate the levels of adenosine at the surface of the monolayer, because adenosine is converted to inosine by adenosine deaminase in the medium. This 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 cardiac fibroblasts were treated with exogenous adenosine, close to basal levels were recovered in the medium (Fig 3⇑). However, when adenosine was added to cardiac fibroblasts pretreated with EHNA, a dramatic increase in adenosine was observed in the medium (Fig 3⇑). Because serum is prepared by clotting blood and red blood cells are well endowed with adenosine deaminase,5 it is possible that a substantial amount of adenosine deaminase is present in the serum and metabolizes adenosine to inosine. Another possibility for the uneven effects of IDO, EHNA, and DIP on cell number is that these drugs have additional effects on cell proliferation not mediated via adenosine.
Can our in vitro findings be extrapolated to physiological situations in vivo, and can adenosine prevent cardiac fibroblast growth in vivo? Our finding that low concentrations of adenosine were able to inhibit cardiac fibroblast growth in the presence but not the absence of EHNA suggests that although adenosine effectively inhibits cardiac fibroblast growth, its effects are underestimated in the present series of experiments, because they were conducted in the presence of FCS, which contains adenosine deaminase. Under physiological conditions in vivo, most of the adenosine deaminase is localized within cells,5 and adenosine in the extracellular compartment will be available in active form to mediate the physiological inhibitory effects on cardiac fibroblast growth. In addition, because adenosine is synthesized by cardiac fibroblasts,29 myocytes,6 and endothelial cells9 10 via multiple pathways, this would ensure pharmacologically active steady-state levels of adenosine locally at the interface between endothelial and cardiac fibroblasts as well as myocytes and cardiac fibroblasts. In contrast, pathological conditions associated with decreased adenosine synthesis or increased adenosine deaminase leakage would reduce the pharmacologically active levels of adenosine, and this would result in decreased antigrowth effects of adenosine. Indeed, data from our laboratory suggest that adenosine deaminase may participate in at least two disease states associated with increased risk of cardiovascular disease, ie, sickle cell anemia and aging/hypertension (unpublished observations).5 However, future studies are needed to confirm or deny this role of adenosine deaminase.
In conclusion, we provide evidence that cardiac fibroblasts synthesize adenosine and that both exogenous and cardiac fibroblast–derived adenosine inhibit FCS-induced growth of ventricular and atrial cardiac fibroblasts in an autocrine fashion. Our findings suggest that adenosine produced by cardiac fibroblasts may play a vital role as a local antigrowth agent and that abnormal/decreased synthesis of adenosine by cardiac fibroblasts or increased catabolism of adenosine by adenosine deaminase or adenosine kinase may contribute importantly to the abnormal growth of cardiac fibroblasts observed in hypertension, myocardial infarction, and reperfusion injury after ischemia. Agents that elevate endogenous adenosine could be clinically important in preventing abnormal growth and proliferation of cardiac fibroblasts in cardiac diseases, thus exerting beneficial effects on the structure of the heart.
Selected Abbreviations and Acronyms
|CGS21680||=||2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamino adenosine hydrochloride|
|HPLC||=||high-performance liquid chromatography|
|SMC||=||smooth muscle cell|
This work was supported by grants from the National Institutes of Health (HL-55314 and HL-35909).
- Received April 21, 1997.
- Accepted May 23, 1997.
- Copyright © 1997 by American Heart Association
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