Mast Cell–Mediated Stimulation of Angiogenesis
Cooperative Interaction Between A2B and A3 Adenosine Receptors
Adenosine is released during tissue injury, ischemia and tumor growth, and promotes angiogenesis. Because mast cells accumulate in the proximity of new blood vessel development, we examined if they may contribute to adenosine-induced angiogenesis. We found that HMC-1 human mast cells express A2A, A2B, and A3 adenosine receptors. The adenosine agonist NECA (100 μmol/L) increased interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 mRNA expression. NECA-induced secretion of IL-8 and VEGF was verified by ELISA. A2B receptors mediate VEGF and IL-8 secretion because neither CGS21680 (selective A2A agonist) nor IB-MECA (selective A3 agonist) produced this effect, and it was inhibited by the selective A2B antagonist IPDX but not by the selective A2A antagonist SCH58261 or the selective A3 antagonist MRS1191. In contrast, the selective A3 agonist IB-MECA (EC50 1 nmol/L) stimulated angiopoietin-2 expression. Conditioned media from NECA-activated HMC-1 stimulated human umbilical vein endothelial cell proliferation and migration, and induced capillary tube formation. Capillary formation induced by mast cell–conditioned media was maximal if both HMC-1 A2B and A3 receptors were activated, whereas activation of A2B receptor alone was less effective. Thus, adenosine A2B and A3 receptors act in a functional cooperative fashion to promote angiogenesis by a paracrine mechanism involving the differential expression and secretion of angiogenic factors from human mast cells.
The purine nucleoside adenosine is an intermediate product of adenine nucleotide metabolism. In many organs adenosine serves as a “retaliatory metabolite” in situations when oxygen supply is decreased or energy consumption is increased. Under these conditions, adenosine is released into the extracellular space and signals to restore the balance between energy supply and local energy requirements. Energy supply to the affected tissue can be modulated acutely by regulation of vascular tone or chronically by formation of new capillaries from preexisting blood vessels, by the process known as angiogenesis. Adenosine, produced in high concentrations during tissue injury, ischemia, or tumor growth, has been implicated in promoting angiogenesis.1–4
There is also growing evidence that mast cells, because of their ubiquitous distribution and location in proximity to blood vessels, can be involved in regulation of angiogenesis. Their number is increased in situations associated with angiogenesis and remodeling, such as arteriosclerosis,5 asthma,6 psoriasis,7 wound repair,8 and tumor growth (see review9). Accumulation of mast cells at the periphery of carcinomatous nodules occurs before the onset of angiogenesis.10 Mast cells can serve as a rich source of cytokines and growth factors that induce or modulate angiogenesis. A widely used human mast cell line HMC-1 has been reported to produce the potent proangiogenic factors interleukin-8 (IL-8)11 and vascular endothelial growth factor (VEGF)12 in response to nonspecific stimulation with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187.
We have previously shown that HMC-1 express functional A2A and A2B receptors, but only A2B receptors induced the production of IL-8.13 In the present study, we determined that, in addition to A2A and A2B receptors, human mast cells express also A3 adenosine receptors. Further experimentation revealed that activation of A2B adenosine receptors stimulates the synthesis of the proangiogenic factors VEGF and IL-8, whereas activation of A3 receptor induces the expression of angiopoietin-2. Stimulation of both A2B and A3 receptors was essential for inducing angiogenesis in a human umbilical vein endothelial cell (HUVEC) model in vitro. We propose that adenosine-induced release of proangiogenic factors from mast cells may represent a novel paracrine mechanism of regulation of angiogenesis in various physiological and pathological situations associated with elevated extracellular concentrations of adenosine. This phenomenon takes advantage of a novel cooperative interaction between A2B and A3 adenosine receptors, which are coexpressed in human mast cells.
Materials and Methods
Human mast cells (HMC-1), a generous gift from Dr J.H. Butterfield (Mayo Clinic, Rochester, Minn), were maintained in suspension culture at a density between 3 and 9×105 cells/mL by dilution with Iscove’s medium supplemented with 10% (vol/vol) FBS, 2 mmol/L glutamine, antibiotics, and 1.2 mmol/L α-thioglycerol.
HUVECs propagated from pooled primary cultures of human umbilical veins were kindly provided by Dr D.E. Vaughan, Vanderbilt University (Nashville, Tenn). HUVECs were maintained in M-199 medium supplemented with 15% (vol/vol) FBS, 1× antibiotic-antimycotic mixture (Gibco BRL catalog No. 15240-062), and 0.3 μg/mL bovine hypothalamus endothelial mitogen (Biomedical Technologies, Stoughton, Mass). All cells were kept under humidified atmosphere of air/CO2 (19:1) at 37°C.
CGS21680 (4-((N-ethyl-5′-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid), NECA (5′-N-ethylcarboxamidoadenosine), and MRS1191 (3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate) were purchased from Research Biochemicals (Natick, Mass). IB-MECA (N6-(3-iodobenzyl)-N-methyl-5′-carbamoyladenosine) was obtained from Tocris Cookson (Ellisville, Mo). [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide), 2200 Ci/mmol, was purchased from NEN Lifescience Products (Boston, Mass). SCH58261 (5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]-pyrimidine) was a generous gift from Drs C. Zocchi and E. Ongini (Schering Plough Research Institute, Milan, Italy). IPDX (3-isobutyl-8-pyrrolidinoxanthine) was synthesized as previously described.14
Gene Expression Assay
Total RNA was isolated using RNeasy mini kit (Qiagen). Expression of angiogenic factors was evaluated using gene expression arrays (Super Array). Human adenosine receptors gene expression array was custom designed by Super Array. The assay was performed according to manufacturer’s instructions.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
VEGF and angiopoietin-2 mRNA expression was determined with dual-quantitative RT-PCR kits (Maxim Biotech) in accordance with manufacturer’s instructions.
Determination of IL-8 and VEGF Levels in Conditioned Media
IL-8 and VEGF concentrations were measured using ELISA kits (R&D Systems) as previously described.13
Western Blot Analysis of Angiopoietin-2 Expression
HMC-1 lysates were separated on 4% to 12% gradient SDS-PAGE gel and processed for Western blotting. Rabbit polyclonal anti-human angiopoietin-2 antibody (Zymed Laboratories, South San Francisco, Calif), 1 μg/mL, was used as a primary antibody, and horseradish peroxidase–conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa) was used as a second antibody at a dilution 1:25 000.
Capillary Tube Formation Assay
To determine morphogenic effects of HMC-1 conditioned media, we used the In Vitro Angiogenesis assay kit (Chemicon). Plates (96-well) were coated with ECMatrix according to the manufacturer’s recommendations. HUVECs were harvested and resuspended in HMC-1 conditioned media. The same media, incubated in the absence of HMC-1, was used for control purposes. HUVECs were seeded at a density of 104 cells/well and incubated for 4 hours under humidified atmosphere of air/CO2 (19:1) at 37°C. Tube formation was inspected under inverted phase-contrast light Olympus IX-70 microscope equipped with a digital camera.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Expression of Adenosine Receptors in Human Mast Cells
Gene expression array results indicate that HMC-1 express mRNA encoding A2A, A2B, and A3 receptors (Figure 1A). No A1 receptor mRNA was detected. Expression levels of A2A, A2B, and A3 receptors were 0.9±0.16% (mean±SEM), 1.97±0.18%, and 0.75±0.05% of β-actin expression, respectively (Figure 1B). These data agree well with previous results of functional assays indicating the presence of A2A and A2B receptors in HMC-1.13,15 In this study, we found that HMC-1 express A3 receptor mRNA in addition to A2A and A2B receptor mRNA. Therefore, we thought it important to confirm the expression of A3 receptor protein.
We used [125I]AB-MECA binding to membranes isolated from HMC-1 to determine the expression of A3 receptor protein (online Figure 1A). Nonlinear regression analysis revealed a Kd of 0.55 nmol/L for [125I]AB-MECA, in agreement with previously published data for human A3 receptors.16 Western analysis of A3 receptor expression in HMC-1 membranes (online Figure 1B) detected immunoreactive bands with apparent molecular masses of 37 and 40 kDa (36 184.6, theoretical Mr for the unglycosylated A3 receptor).
We also tested if A3 receptors are linked to the same intracellular signaling pathways regulated by A2A and A2B adenosine receptors in HMC-1. Online Figure 2A shows that the nonselective adenosine agonist NECA, acting on both A2A and A2B adenosine receptors, stimulates adenylate cyclase more efficaciously than the selective A2A agonist CGS21680, in agreement with previous reports.13,15 The specific A3 agonist IB-MECA failed to inhibit forskolin-stimulated adenylate cyclase at concentrations (10−9 to 10−7 mol/L) that selectively activate A3 receptors (online Figure 2B). Therefore, we found no evidence for functional coupling of A3 receptors to adenylate cyclase in HMC-1. Similarly, only the nonselective adenosine agonist NECA stimulated phosphoinositol hydrolysis, whereas the selective A2A agonist CGS21680 and the selective A3 agonist IB-MECA had no effect (online Figure 3). These data confirm that only A2B receptors are functionally coupled to stimulation of phospholipase Cβ in HMC-1.
Adenosine-Induced Expression of Angiogenic Factors in Human Mast Cells
Incubation of HMC-1 in the presence of 100 μmol/L NECA and 1 U/mL adenosine deaminase for 3 hours increased mRNA expression of the angiogenic factors IL-8, VEGF, and angiopoietin-2 by 9.3±0.4-, 3.0±0.1-, and 2.2±0.4-fold, respectively, compared with cells incubated with vehicle and 1 U/mL adenosine deaminase. After 6 hours of incubation with NECA, the increase in IL-8 and VEGF mRNA expression was on the decline (3.4±0.5- and 1.8±0.2-fold), whereas the expression of angiopoietin-2 increased further, to 7.7±0.7-fold (Figure 2A). Figure 2B illustrates this differential kinetics of adenosine-induced expression of angiogenic factors. The expression of other genes in our array was either negligible (<0.1% of β-actin expression) or was not changed (<2-fold) on stimulation with NECA. The list of genes included in this assay can be found in the expanded Materials and Methods section of the online supplement.
Adenosine Stimulates IL-8 Production via A2B Receptors
Measurements of IL-8 protein levels in conditioned media confirmed the results of gene array analysis. NECA produced a dose-dependent increase in IL-8 levels in conditioned media after 6 hours of incubation with HMC-1, from 6.1±1.8 to 97.8±3.8 pg/mL. The selective A2B antagonist IPDX competitively inhibited NECA-induced IL-8 secretion, indicating that this process is mediated via A2B receptors (Figure 3A). We showed previously that stimulation of A2A adenosine receptors had no effect on IL-8 production in HMC-1.13 As seen in Figure 3B, the selective A3 agonist IB-MECA did not stimulate IL-8 production and the selective A3 antagonist MRS1191 did not inhibit the IL-8 production induced by NECA.
Adenosine Increases VEGF Expression via A2B Receptors
We used dual-quantitative RT-PCR to confirm adenosine-dependent increase in VEGF mRNA expression and also to determine the effect of adenosine on the expression of the major VEGF isoforms VEGF121, VEGF165, and VEGF189. Amplification of cDNA using a specific set of primers common to all three VEGF isoforms showed the increase in their expression in HMC-1 stimulated with 100 μmol/L NECA for 3 hours compared with unstimulated cells (Figures 4A and 4B). The increase ranged from 1.23-fold for VEGF189 to 3.2-fold for VEGF121.
HMC-1 tonically release VEGF (Figure 4C). VEGF secretion was increased in the presence of NECA. VEGF concentrations in conditioned media from HMC-1 incubated for 6 hours in the absence or in the presence of 100 μmol/L NECA were 1.21±0.09 and 2.09±0.08 ng/mL, respectively. As seen in Figure 4D, only the nonselective agonist NECA stimulated VEGF production. Neither the selective A2A agonist CGS21680 nor the selective A3 agonist IB-MECA increased VEGF levels. Furthermore, only the selective A2B antagonist IPDX, but not the selective A2A antagonist SCH58261 or the selective A3 antagonist MRS1191, inhibited the NECA-stimulated VEGF secretion. Based on these data, we concluded that A2B receptors mediate VEGF increase in HMC-1.
Adenosine Induces Angiopoietin-2 Expression via A3 Receptors
As shown above, NECA stimulated angiopoietin-2 expression with kinetics different from those for stimulation of IL-8 and VEGF (Figure 2B). Angiopoietin-2 expression also was different from IL-8 and VEGF by its sensitivity to the A3 selective adenosine agonist IB-MECA. As seen in Figures 5A and 5B, IB-MECA increased angiopoietin-2 mRNA expression with an EC50, 1.2 nmol/L, that agrees with its reported affinity at human A3 receptors.16 Furthermore, only the selective A3 antagonist MRS1191, but not the selective A2A antagonist SCH58261 or the selective A2B antagonist IPDX, inhibited the NECA-stimulated angiopoietin-2 expression (online Figure 4).
Angiopoietin-2 protein expression was demonstrated in lysates of HMC-1 stimulated with 100 μmol/L NECA for 12 hours. Figure 5C shows angiopoietin-2 immunoreactivity detected in lysates of stimulated HMC-1 (lanes 6 and 7) but not in lysates of unstimulated cells (lanes 4 and 5).
Effects of HMC-1 Conditioned Media on Angiogenesis In Vitro
Conditioned media, collected from HMC-1 exposed to 10 μmol/L NECA for 48 hours, stimulated HUVEC proliferation by 25±9% (n=6) (online Figure 5) and migration by 103±5% (n=4) (online Figure 6), compared with conditioned media from unstimulated HMC-1.
Conditioned media collected from NECA-stimulated HMC-1 promoted capillary tube formation of HUVECs incubated on ECMatrix for 4 hours. HUVECs, incubated with conditioned media from unstimulated HMC-1, showed only few cells aligned in an organized manner, with the majority of them remaining unassociated (Figures 6A and 6C). In contrast, conditioned media from NECA-stimulated HMC-1 induced the formation of a characteristic tube network (Figures 6B and 6D). In parallel experiments, we found that nonconditioned medium containing 10 μmol/L NECA did not have a direct angiogenic action on HUVECs. The effects of conditioned media on HUVEC tube formation were inhibited when HMC-1 were incubated with NECA in the presence of the selective A2B antagonist IPDX 10 μmol/L (Figure 6E) and, to a lesser extent, in the presence of the selective A3 antagonist MRS1191 100 nmol/L (Figure 6F). In ancillary studies, we demonstrated that this effect cannot be explained by cytotoxic effects of adenosine antagonists, because nonconditioned media containing 10 μmol/L IPDX or 100 nmol/L MRS1191 did not affect viability of HUVECs (data not shown). Furthermore, conditioned media from HMC-1 stimulated with the selective A3 agonist IB-MECA did not induce HUVEC capillary formation (Figure 7) indicating that stimulation of A3 receptor alone is not sufficient to induce angiogenesis in vitro.
Treatment of conditioned media from NECA-stimulated HMC-1 with neutralizing antibodies specific for VEGF abolished its ability to induce HUVEC tube formation, indicating the importance of VGEF in our model. Antibodies against IL-8 failed to inhibit HUVEC tube formation, probably reflecting the relatively low concentrations of IL-8 achieved in our model (online Figure 7).
The evidence pointing to an association between mast cells and angiogenesis has been growing for more than a hundred years. Shortly after the discovery of mast cells, it was recognized that they tend to concentrate around blood vessels in inflammatory and neoplastic foci.17 It was later demonstrated that mast cells accumulate in the proximity of tumors before the onset of tumor-associated angiogenesis.10 Their numbers also rise in other physiological and pathological events associated with an increase in angiogenic activity.5,6,8 However, it is unclear which stimuli would signal mast cells to induce neovascularization. The events leading to angiogenesis are often accompanied by tissue hypoxia and the subsequent increase in extracellular adenosine. For example, adenosine is released into extracellular space at high concentrations at early stages of solid tumor growth due to severe hypoxia and necrosis. Tissue injury, ischemia, and inflammation are also accompanied by an increase in extracellular adenosine concentrations. We and others have shown that adenosine stimulates the secretion of the angiogenic factors IL-8, VEGF, and basic fibroblast growth factor (bFGF) from microvascular endothelial cells.1–4 In this study, we tested the hypothesis that extracellular adenosine can promote angiogenesis by triggering the synthesis and release of angiogenic factors from mast cells.
We have previously reported the presence of functional A2A and A2B adenosine receptors in the human mast cell line HMC-1.13 In the present study, we found that HMC-1 express also A3 receptor mRNA in addition to A2A and A2B transcripts. Radioligand binding experiments and Western blot analysis confirmed the expression of A3 receptor protein on the surface of HMC-1. A3 receptors are not linked to adenylate cyclase or phospholipase Cβ, the intracellular signaling systems regulated by A2B receptors in HMC-1. Although coupling of A3 to adenylate cyclase via αi and to phospholipase Cβ via βγ subunits has been shown in some cells, recent studies in ventricular myocytes demonstrated that A3 receptors are coupled to RhoA and phospholipase D.18,19 The intracellular signaling pathways linked to A3 receptors in HMC-1 remain to be elucidated.
There is growing evidence that IL-8 plays an important and specific role in promoting angiogenesis.20–22 IL-8 is elevated in wounds and enhances wound healing.23 IL-8, secreted by inflammatory and neoplastic cells, stimulates angiogenesis in various solid tumors.20–22 We have previously reported that A2B, but not A2A, receptors stimulate IL-8.13 In this study, we found that A3 receptors are not involved in IL-8 secretion either; the selective A3 agonist IB-MECA failed to stimulate IL-8 secretion, and NECA-induced IL-8 secretion was competitively inhibited by the selective A2B antagonist IPDX but not by the selective A3 antagonist MRS1191. Of interest, while this article was in preparation, Meade et al24 reported the presence of A3 receptors on HMC-1 and the failure of these receptors to stimulate IL-8 release. Furthermore, they found that A2B-mediated IL-8 release could be greatly potentiated by stem cell factor. Taken together, these results confirm that A2B receptors are solely responsible for adenosine-induced secretion of IL-8.
In this study, we found that adenosine A2B receptors also stimulate the production of another potent angiogenic factor, VEGF. Mast cells were known to produce VEGF,25–28 and HMC-1 were found to constitutively produce and secrete VEGF in vitro.12 We demonstrate, for the first time, that VEGF mRNA expression and protein secretion are specifically stimulated by adenosine in human mast cells. Adenosine preferentially increases mRNA expression of VEGF121 and VEGF165, the diffusible isoforms that are secreted in the medium. Adenosine increased to a lesser extent mRNA expression of VEGF189, the isoform that is mostly bound to heparin-containing proteoglycans of the extracellular matrix. This pattern of stimulation of VEGF isoforms by adenosine correlates with the increase in VEGF secretion. NECA stimulated VEGF secretion from HMC-1 by ≈1.7-fold over its constitutive secretion. Using selective agonists and antagonists, we determined that A2B adenosine receptors regulate VEGF production in human mast cells, in addition to IL-8. Our results, therefore, suggest that angiogenesis is an important function of adenosine A2B receptors in mast cells.
In addition to upregulation of IL-8 and VEGF secretion, we also found that adenosine stimulates the expression of angiopoietin-2 in human mast cells. The difference between the time course of stimulation of mRNA expression of angiopoietin-2, and that of IL-8 and VEGF, led us to explore the possibility that their expression is regulated by distinct receptors. Indeed, our results demonstrate that, whereas A2B receptors mediate the expression of IL-8 and VEGF, angiopoietin-2 expression is mediated by A3 adenosine receptors. To the best of our knowledge, adenosine has not been previously shown to release angiopoietin-2. Our results raise the possibility that adenosine can contribute to angiogenesis not only by stimulation of VEGF and IL-8 secretion via A2B receptors but also by stimulation of angiopoietin-2 secretion via A3 receptors.
Localization of angiopoietin-2 primarily at sites of vascular remodeling suggests its role in neovascularization. It has been proposed that angiopoietin-2 participates in angiogenesis by blocking the stabilizing action of angiopoietin-1 on endothelial cells. According to this generally accepted model, angiopoietin-2 is viewed as an enhancer of angiogenic signals. Angiopoietin-2 does not induce angiogenesis on its own, but primes blood vessels for angiogenesis and facilitates the effects of VEGF.29 Based on our observations, we suggested that such a model of cooperative regulation of angiogenic response could be applied to regulation of mast cell–mediated angiogenesis by adenosine. In this model (Figure 8), A3 receptors on mast cells would play the role of an “enhancer” of angiogenic signals mediated by A2B receptors. During tumor growth, tissue injury, ischemia, or inflammation, adenosine is released into the extracellular space. Binding of adenosine to mast cell A2B receptors stimulates the secretion of VEGF, IL-8, and possibly other angiogenic factors. The effect of VEGF on new capillary formation is facilitated by the concomitant stimulation of mast cell A3 receptors that induce the expression of angiopoietin-2. This model was validated in experiments with HUVEC tube formation. Our results indicate that A2B and A3 receptors interact in a functional cooperative fashion to promote angiogenesis. This conclusion is based on the observation that tube formation was substantially greater when human endothelial cells were exposed to conditioned media from human mast cells if both A2B and A3 receptors were simultaneously activated compared with the selective activation of A2B receptor alone. Such cooperation between adenosine receptors is a novel finding. Adenosine A2B receptors appear to be ubiquitously expressed, but in virtually every cell they are coexpressed with other adenosine receptor subtypes. The functional relevance of this coexpression had not previously been investigated. There is currently no evidence that a similar cooperation exists between A2B and A3 receptors in the modulation of mast cell–mediated inflammatory processes, but such a possibility should be explored.
It should be noted that our experiments were done in an established mast cell line HMC-1 with phenotypic characteristics resembling MCT-type human mast cells.30 Mast cells are heterogeneous, and not all mast cell lines may express the same adenosine receptor subtypes as HMC-1. Similarly, we chose HUVECs as a model to study paracrine regulation of angiogenesis because these cells do not secrete VEGF, IL-8, or angiopoietin-2 in response to adenosine.4 However, endothelial cells are also heterogeneous. We have previously shown that A2B receptors can directly induce release of VEGF and IL-8 from microvascular endothelial cells.2,4 It is possible that in some vascular beds adenosine provides autocrine stimulation of angiogenesis by acting directly on endothelial A2B receptors, in addition to paracrine modulation mediated by mast cell A2B and A3 receptors. Finally, we cannot exclude that, in addition to VEGF, IL-8, and angiopoietin-2, there are also other factors involved in the angiogenic response induced by adenosine. For example, mast cells were shown to stimulate angiogenesis by releasing tryptase, chymase, and matrix metalloproteinase-9 (MMP-9).31–33 In this study, we screened only a limited number of angiogenesis-related genes in human mast cells. Nonetheless, our findings that adenosine stimulates secretion of such angiogenic factors as VEGF and IL-8 via A2B receptors, and angiopoietin-2 via A3 receptors, demonstrate for the first time an example of functional cooperation between different adenosine receptor subtypes in promoting an angiogenic response.
In summary, we have shown the presence of A2B and A3 receptors in human mast cells. Activation of A2B receptors leads to the secretion of VEGF and IL-8, whereas activation of A3 receptors upregulates the expression of angiopoietin-2. These, and potentially other factors released by mast cells, can act synergistically, and in a paracrine fashion, on endothelial cells to induce angiogenesis.
This work was supported by NIH grants R29 HL55596 and R01 HL70073 (to I.F.) and R01 HL67232 (to I.B.).
- Received October 17, 2002.
- Revision received January 16, 2003.
- Accepted January 30, 2003.
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