Cellular Biology |
From the Departments of Medicine (M.B.G., R.W.T., S.C., M.I.D., P.E.S., J.C.S., L.B.), Ophthalmology (M.B.G.), and Pharmacology and Therapeutics (M.J.O., J.C.S., L.B.), University of Florida, Gainesville, Fla, and Department of Medicine and Pharmacology (I.F., I.B.), Vanderbilt University, Nashville, Tenn. L.B.'s current address is Pharmacological Sciences, CV Therapeutics, Palo Alto, Calif.
Correspondence to M.B. Grant, MD, Associate Professor of Medicine, Department of Medicine, Division of Endocrinology and Metabolism, University of Florida, P.O. Box 100226, Gainesville, FL 32610-0226. E-mail grantma{at}medicine.ufl.edu
| Abstract |
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Key Words: adenosine receptor angiogenesis ischemia hypoxia diabetes
| Introduction |
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Adenosine, the subject of the present study, has been proposed to be a factor that links altered cellular metabolism caused by oxygen deprivation to the formation of new capillaries.7 8 9 This proposed role of adenosine is based on the observation that this nucleoside is released in increased amounts by hypoxic and/or ischemic cells and promotes proliferation of endothelial cells.9 10 11 Consistent with this hypothesis, adenosine and adenosine analogs have been reported to affect a number of steps involved in angiogenesis, including endothelial cell proliferation,8 12 13 14 migration,12 15 16 and blood vessel formation in various vascular beds.15 17 Adenosine can interact with at least 4 subtypes of G proteincoupled receptors, designated A1, A2A, A2B, and A3.18 These receptor subtypes are encoded by distinct genes and can, for the most part, be differentiated on the basis of their affinities for selected agonists and antagonists.19 20 A1 and A3 adenosine receptors are coupled to pertussis toxinsensitive inhibitory G proteins that inhibit adenylyl cyclase activity, whereas A2A (high-affinity) and A2B (low-affinity) adenosine receptors are coupled to cholera toxinsensitive G proteins that stimulate adenylyl cyclase activity.21 In most cell types and organ systems, activation of A1 adenosine receptors results in decreased work, and therefore, reduced O2 consumption. Activation of A2A adenosine receptors, on the other hand, increases O2 supply by causing vasodilation.22 Thus, adenosine is an ideal metabolite to respond to imbalances between O2 supply and demand. In the retina, hypoxia is followed by compensatory angiogenesis, which is detrimental and results in aberrant blood vessels that are friable and prone to bleeding.23
VEGF is a potent endothelial mitogen, induced by hypoxia and hyperglycemia, and has been shown to be an important factor in ischemic ocular neovascularization.24 25 26 VEGF causes hyperpermeability of blood vessels, which is observed in both nonproliferative and proliferative diabetic retinopathy. VEGF acts through 2 receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), both of which are tyrosine kinases. VEGF signaling occurs through tyrosine phosphorylation of phospholipase C (PLC) and phosphatidylinositol 3'-kinase.27 28 The effects of VEGF are also mediated by activation of protein kinase C (PKC) to induce membrane translocation of PKC isoforms, especially the ß-isoform of the enzyme.25 VEGFR-1 mediates the permeability effects associated with VEGF, whereas VEGFR-2 mediates the proliferative effects of VEGF. Elevated levels of VEGF have been detected in vitreous humor of diabetic patients with proliferative retinopathy.25 More importantly, in animal models of retinal neovascularization, inhibition of VEGF blocks neovascularization.29 Other growth factors have been implicated in ocular angiogenesis, including bFGF30 and IGF-I.31
The experiments described were performed test the hypothesis that adenosine regulates expression of the angiogenic growth factor VEGF and to determine the adenosine receptor subtype that mediates the effect of the nucleoside in retinal endothelial cells of human origin.
| Materials and Methods |
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Human retinal endothelial cells (HRECs) were prepared and maintained as previously described.33 Cells in passages 3 to 6 were used for the studies. The identity of HRECs was validated by demonstrating endothelial cell incorporation of fluorescently labeled acetylated LDL and by fluorescence-activated cell sorting analysis as previously described.33 For all experiments, cells were starved of serum overnight and then incubated with adenosine deaminase type III (2 U/mL, Sigma-Aldrich) for 20 minutes before test agents were added. Adenosine receptor agonists and antagonists were added at concentrations ranging from 5 nmol/L up to 100 µmol/L in serum-free medium containing adenosine deaminase type III and then incubated for additional times as indicated in specific results.
cAMP was measured in response to adenosine receptor agonists and/or antagonists as described.34 Conditioned medium was used to measure changes in VEGF protein in response to adenosine receptor agonists and/or antagonists using an ELISA kit (R&D Systems, Inc). HREC proliferation was determined by measuring DNA synthesis via colorimetric detection of bromodeoxyuridine (BrdU) incorporation using a kit (Roche Molecular Biochemicals), and also by changes in cell number. BrdU incorporation was also used to measure the effect of anti-VEGF antibody on adenosine receptor agonistinduced HREC proliferation. The potential reduction of VEGF synthesis induced by adenosine receptor agonist was tested by ELISA after inclusion of antisense oligonucleotides directed against mRNA for either A2B adenosine receptor or VEGF. Quantitative reverse transcriptasepolymerase chain reaction (RT-PCR), using a competitive synthetic multiplex template as described previously,35 was performed to measure changes in mRNA after treatment with adenosine receptor agonists and/or antagonists. Immunofluorescent confocal microscopy was used as described36 to demonstrate both the presence of A2B adenosine receptors and uptake of fluorescence-labeled acetylated LDL in HRECs grown on multichamber glass slides (Nalge Nunc International).
Statistical Analysis
Comparisons between treatment groups (as described in the figure
legends) were analyzed by 1-way ANOVA followed by the
Bonferroni t test. Data are expressed as mean±SEM. Values
of P<0.05 were considered statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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The data for cell counts were consistent with those for BrdU
incorporation. Treatment with NECA for 48 hours resulted in a
concentration-dependent increase in HREC number, whereas neither
CGS21680 nor CPA caused an increase in cell number (Figure 2A
). Of the 3 adenosine receptor
antagonists tested, only XAC (10 µmol/L)
significantly inhibited the increase in cell number induced by 10
µmol/L NECA (Figure 2B
).
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cAMP Accumulation
To obtain evidence for the presence of the
A2B adenosine receptors in HRECs, we
performed assays for cAMP content in intact HRECs after treatment of
cells with adenosine receptor agonists and
antagonists. The nonselective adenosine receptor
agonist NECA increased the cAMP content of HRECs in a
concentration-dependent manner (Figure 3A
), with an EC50
value of 24 µmol/L. In contrast, the selective high-affinity
A2A adenosine receptor agonist CGS21680
(at concentrations up to 100 µmol/L) had no significant effect
on cAMP content of HRECs (Figure 3A
). The effect of selective
A1 and A2A
adenosine receptor antagonists on NECA-induced
accumulation of cAMP was also examined. NECA (10
µmol/L)-induced increase in cAMP content in HRECs was not
significantly inhibited either by the selective
A2A adenosine receptor
antagonist SCH58261 (60 nmol/L) or by the selective
A1 adenosine receptor
antagonist CPX (20 nmol/L) (Figure 3B
). On the other
hand, the nonselective adenosine receptor
antagonist XAC (10 µmol/L) completely blocked the
effect of NECA on cAMP accumulation.
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Quantification of VEGF, IGF-I, and bFGF mRNA in HRECs
To determine whether NECA stimulates angiogenic growth factor mRNA
expression in HRECs, total mRNA from HRECs exposed to NECA was
subjected to quantitative RT-PCR. Treatment of HRECs with NECA (10
nmol/L to 10 µmol/L) for 2 hours induced a
concentration-dependent increase in expression of mRNA for VEGF by up
to 4.6-fold (from 0.4x106 to
1.85x106 copies/µg RNA), compared with
untreated control cells. After 8 hours of exposure to NECA, cell mRNA
levels for VEGF in cells treated with NECA had returned to baseline.
HRECs were also treated with the A2A
adenosine receptor agonist, CGS21680 (10 nmol/L to 10
µmol/L), and the A1 adenosine receptor
agonist, CPA (10 nmol/L to 10 µmol/L). In contrast to NECA,
neither CGS21680 nor CPA caused a significant change in VEGF mRNA
expression (data not shown).
The increase in mRNA for VEGF caused by 10 µmol/L NECA was not
attenuated significantly either by the selective
A2A adenosine receptor
antagonist SCH58261 (60 nmol/L) or by the selective
A1 adenosine receptor
antagonist CPX (20 nmol/L) (Figure 4
). The nonselective
antagonist XAC (10 µmol/L) completely attenuated
NECA-induced increases in mRNA for VEGF (Figure 4
). NECA
(10 µmol/L) also induced a time-dependent increase in mRNA for
both IGF-I and bFGF. IGF-I mRNA levels increased by 2.2-fold after 2
hours of exposure (from 30x103 copies/µg RNA
to 65x103 copies/µg RNA) and 11.7-fold
(350x103 copies/µg RNA) after 8 hours of
exposure to NECA. Similarly, bFGF mRNA increased 3.7-fold after 2 hours
of exposure (from 2x103 copies/µg RNA to
7.4x103 copies/µg RNA) and 11.4-fold
(22.8x103 copies/µg RNA) after 8 hours of
exposure to NECA.
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Quantification of VEGF Protein in Conditioned Medium
To determine whether the increase in VEGF mRNA expression resulted
in increased protein levels, VEGF was measured in conditioned medium
after 8 hours of exposure to NECA, in the presence or absence of
adenosine receptor antagonists (Figure 5
). NECA increased VEGF protein, but
neither the A1 adenosine receptor agonist
CPA (not shown) nor the A2A adenosine
receptor agonist CGS21680 caused an increase in VEGF protein (Figure 5
). The increase in VEGF protein caused by NECA was not
attenuated by either the selective A2A
adenosine receptor antagonist SCH58261 (60 nmol/L)
or by the selective A1 adenosine receptor
antagonist CPX (20 nmol/L). Only the nonselective
adenosine receptor antagonist XAC (10
µmol/L) completely inhibited the action of NECA to increase VEGF
protein expression (Figure 5
).
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Effect of Anti-VEGF Antibody on NECA-Induced HREC
Proliferation
Incubation with 10 ng/mL VEGF resulted in BrdU incorporation to a
level approximating that induced by normal growth medium. The anti-VEGF
antibody at 100 ng/mL significantly reduced DNA synthesis induced by
VEGF (Figure 6
). Incubation with NECA
(10 µmol/L) increased DNA synthesis to levels comparable with
that induced by normal growth medium. The addition of anti-VEGF
antibody resulted in a decrease in NECA-induced BrdU incorporation,
which was statistically significant at the highest concentration of
antibody used (Figure 6
). Similar results were observed at
either 24 or 48 hours of exposure to the test agents.
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Effect of Antisense Oligonucleotides on VEGF
Induction by NECA
Both A2B adenosine receptor
and VEGF antisense oligonucleotides caused a
significant decrease of VEGF in the conditioned medium after NECA
exposure (Figure 7
). This effect was most
pronounced for the receptor antisense oligonucleotide
with 10 nmol/L NECA, but it was evident for all concentrations of NECA
tested. The VEGF antisense oligonucleotide also caused
a decrease in secreted VEGF in response to NECA, although not to the
same magnitude as that observed with the A2B
adenosine receptor antisense.
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Immunofluorescence
Analysis of acetylated LDL uptake indicates
that the cells are indeed of endothelial origin (Figure 8A
). These results were confirmed
by immunofluorescent labeling with antibody to coagulation
factor VIII (data not shown). Labeling with A2B
adenosine receptor antibody clearly demonstrated that the
tested cells express the A2B receptor subtype
(Figure 8B
and 8C
).
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| Discussion |
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In HRECs, NECA caused a concentration-dependent increase in VEGF mRNA as well as an increase in secreted VEGF protein that was blocked by an antisense oligonucleotide complementary to A2B adenosine receptor mRNA. In contrast, neither the A2A agonist CGS21680 nor the A1 agonist CPA affected the expression either of VEGF mRNA or protein, ruling out a role for either A2A or A1 adenosine receptors in mediating increased VEGF expression, increased BrdU incorporation, and cell proliferation. NECA-induced increases in expression of both VEGF mRNA and protein by HRECs were blocked by the nonselective adenosine receptor antagonist XAC, whereas the A1- and A2A-selective adenosine receptor antagonists CPX (20 nmol/L) and SCH58261 (60 nmol/L), respectively, did not attenuate these increases. The antagonists CPX and SCH58261 were used at concentrations at which their selectivity for A1 and A2A receptors has been demonstrated in cardiovascular preparations.18 37 38 On the other hand, XAC was used at a concentration (10 µmol/L) that should be sufficient to antagonize effectively A1, A2A, A2B, and possibly the A3 receptor-mediated responses.
Hence, the evidence supporting the role of A2B adenosine receptors as the adenosine receptor subtype that mediates the effects of NECA reported here can be summarized as follows: (1) A2B receptors were localized in HRECs using immunofluorescence microscopy with the A2B antibody; (2) antisense oligonucleotides homologous to the A2B receptor blocked NECA-stimulated VEGF production; (3) neither the A1 nor the A2A receptor agonists had any effect on BrdU incorporation, cell proliferation, or cAMP production; (4) neither the A1 nor the A2A antagonists, used at the concentration at which they are selective for their receptor subtype, antagonized the effects of NECA; and (5) the nonselective but potent A2B antagonist XAC used at high concentrations significantly attenuated the effects of NECA.
Taken together, the data support the hypothesis that the A2B adenosine receptor, but neither the A1 nor the A2A receptor, is responsible for mediating the actions of NECA on cAMP accumulation and VEGF synthesis in cultured HRECs. The results of our studies do not rule out a possible role of A3 receptor in mediating the effects of NECA. However, this is unlikely, because the affinity (Ki) of XAC for the A3 adenosine receptor is 29 µmol/L, which is higher than the concentration (10 µmol/L) used in our studies.37 39 Our conclusion that the A2B receptor is the most likely adenosine receptor subtype that mediates the effects of NECAand presumably adenosineon HRECs differs from that reported by Takagi et al.40 41 These investigators, using retinal endothelial cells of bovine origin, concluded that the proliferative action of adenosine is mediated by A2A receptor. Takagi et al40 also reported that acute hypoxia causes a decline in KDR/Flk mRNA levels as well as VEGF binding sites on the cell surface. On the other hand, chronic hypoxia was associated with increased KDR/Flk message levels.40 More importantly, Takagi et al41 also reported that the endogenous adenosine released by hypoxic bovine retinal endothelial cells was sufficient to stimulate VEGF message expression. Species differences and passage number may account for differences in the observed adenosine receptor subtype in retinal endothelial cells. Furthermore, distinct adenosine receptor subtypes may mediate the proliferative effects of adenosine in endothelial cells from different vascular beds, even within the same species.
Protein kinase A and members of the mitogen-activated protein kinase, family such as extracellular signalregulated kinases 1 and 2 (ERK1/ERK2) are potential mediators of adenosinemediated cell proliferation.42 43 44 Activation of the A2B adenosine receptor results in cAMP generation via Gs. A2 receptor activation and stimulation of adenylyl cyclase/protein kinase A pathways can either activate11 or inhibit45 46 47 growth factorstimulated ERK activity.
A2B adenosine receptor signaling through Gq/11 also results in increased levels of ERK.42 Therefore, A2B receptor stimulation of Gq/11, PLC, and PKC may synergize with or potentiate the effects of traditional tyrosine kinasecoupled growth factors,48 49 either through c-srcdependent activation of ERK or through PKC-dependent, src-independent, pathways. Because forskolin-mediated adenylyl cyclase activity does not activate VEGF expression in endothelial cells,50 Gq/11- and PKC-mediated activation of ERK may contribute to activation of transcription factors and lead to the induction of message for VEGF.51 Thus, A2B receptor activation can mediate proliferation by inducing growth factor synthesis and through stimulation of Gq/11, PLC, and PKC pathways.
Angiogenesis is a compensatory mechanism in response to insufficient tissue oxygenation.2 In the retina of diabetic individuals, homeostatic abnormalities lead to retinal nonperfusion and subsequent ischemia.52 Ischemia leads to new vessel formation and disruption of the normal retinal vasculature, the hallmarks of proliferative diabetic retinopathy.23 Our findings raise the possibility that selective A2B adenosine receptor antagonists could be used as a novel therapeutic approach to block the inciting events leading to aberrant angiogenesis in proliferative diabetic retinopathy. Pharmacological modulation of the neovascular response in a nondestructive manner should have significant advantages over current therapeutic approaches.
By blocking the A2B adenosine receptor, the action of adenosine to induce the growth factor cascade may be inhibited, and blocking the A2B adenosine receptor may attenuate aberrant cellular proliferation. In summary, our results provide strong evidence that the proliferative effect of adenosine on HRECs is caused by increased expression of VEGF and probably other growth factors, and this effect is mediated by the A2B adenosine receptor.
| Acknowledgments |
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Received December 22, 1998; accepted August 11, 1999.
| References |
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