The A2b adenosine receptor protects against vascular injury

Abstract

The A2b adenosine receptor (A2bAR) is highly abundant in bone marrow macrophages and vascular smooth muscle cells (VSMC). To examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis after angioplasty. While considering related mechanisms, we noted higher levels of TNF-α, an up-regulator of CXCR4, and of VSMC proliferation in the injured KO mice. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is up-regulated in lesions of the KO mice. In addition, aortic smooth muscle cells derived from A2bAR KO mice display greater proliferation in comparison with controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study highlights the significance of the A2bAR in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation.

The cardiovascular protective effects of adenosine have been described in recent years. Although adenosine activates multiple receptor subtypes (A1, A2a, A2b, and A3 receptors), the adenylyl cyclase stimulatory A2a adenosine receptors (A2aARs) are regarded mainly as cardioprotective receptors (1). In general, the A2-type receptors have been described as sensors of tissue damage during an immune response (2), and A2aARs have been described as having a nonredundant role in the attenuation of inflammation and tissue damage in vivo (3). Indirect evidence suggests that the adenylyl cyclase stimulatory A2b adenosine receptors (A2bARs) also affect vascular function (4–6). For instance, adenosine inhibits the proliferation of murine VSMC via activation of the A2bAR (4). On the other hand, A2bAR activation induces growth of human vascular endothelial cells and stimulates MAP kinase activity in other cell types (5, 6). Additional studies suggest that adenosine induces apoptosis of human arterial smooth muscle cells, which is mediated via the A2bAR in a cAMP-dependent pathway (7). Interestingly, an in vivo model of smooth muscle cell proliferation revealed that an A2 receptor agonist, 2-octynyladenosine, inhibited neointima formation (8). Furthermore, it has been reported that A2aAR activation reduces neointimal formation in a carotid ligation injury model (9). Adenosine receptors also play a major role in several inflammation-associated processes by inhibiting immune activation and preventing excessive tissue damage. This has been demonstrated by using A2aAR-null mice (3). To examine whether the low-affinity A2bARs have an effect on vascular phenotypes, we generated an A2bAR KO/reporter gene knockin mouse model (10). Our earlier studies demonstrated that the A2bARs are notably expressed in vascular cells and macrophages and protect against excessive vascular adhesion (10), suggesting that A2bARs mediate a protective effect on the vasculature.

In the current study we examined the role of vascular and bone marrow adenosine receptors in vascular response to injury using a model that mimics human restenosis after angioplasty. We found an important role for this receptor in regulating lesion formation associated with an increased level of the progenitor-attracting protein CXCR4.

Results

A2bAR Deficiency Enhances Postinjury Neointima Formation in the Vasculature.

A guidewire was used to injure the femoral artery of age-, gender-, and strain-matched WT or A2bAR KO mice. Histological examination revealed that the neointima is markedly thickened in A2bAR KO mice compared with WT mice. The average area of the intima as well as the ratio of the intima to the media (I/M) is >2-fold greater in the KO mice than in WT mice (Fig. 1 and Table 1). At 4 weeks after injury, the neointimal cells are largely positive for α-smooth muscle actin, a conventional marker for smooth muscle cells [supporting information (SI) Fig. 6]. A2bAR deficiency has no effect on the basal level of thickness of the vascular wall (SI Fig. 7 A and B).

Fig. 1.

Lesion formation in WT and A2bAR KO mice after injury of the femoral artery. Masson's Trichrome-stained sections of injured and sham-injured femoral arteries evaluated 4 weeks after injury. Red arrows point to the neointimal area. (Magnification: ×200.)

Table 1.

Quantification of the lesions formed in response to femoral artery injury

Mice	Vessel area, μm2	Luminal area, μm2	Intimal area, μm2	Medial area, μm2	I/M ratio
WT (n = 8)	74,132 ± 41,738	49,980 ± 32,811	11,335 ± 6,203	12,822 ± 8,552	1.12 ± 0.7
A2bAR KO (n = 7)	75,861 ± 28,595	39,664 ± 27,008	23,348 ± 14,030	12,848 ± 7,584	2.43 ± 1.18

Quantification of the lesions formed in response to femoral artery injury calculated by the application of Image-Pro Plus analysis software on digitized images. The intima/media (I/M) ratio reflects averages of individual I/M ratios measured for each mouse and not a division of the averaged intimal area by the averaged medial area. Data are expressed as mean area ± SEM for A2bAR KO versus WT mice (WT, n = 8; A2bAR KO, n = 7) using a two-tailed unpaired Student t test. Differences of P ≤ 0.05 were considered statistically significant. Intimal area and I/M ratio in A2bAR KO mice are significantly higher as compared with WT mice (P ≤ 0.05).

Augmented Inflammatory Response Contributes to Neointima Formation in A2bAR KO Mice.

In view of the facts that A2bAR is expressed on macrophages and that the deletion of A2bAR increases the levels of proinflammatory cytokines, such as TNF-α (10), it was of interest to determine the levels of these cytokines after vascular injury in A2bAR KO and WT mice. As shown in Fig. 2A, plasma levels of TNF-α are ≈2-fold greater in noninjured (control) A2bAR KO mice compared with WT mice. At 1 day after injury TNF-α is elevated in all mice, and it is 1.4-fold higher in KO mice as compared with WT (P < 0.05), although the IL-6 level is not significantly different in the two mouse models. However, at 4 weeks after injury, both TNF-α (5.7-fold higher than WT) and IL-6 (2.7-fold higher than WT) levels are increased in KO mice. In accordance with the known effect of TNF-α on recruitment of inflammatory cells (11), leukocytes accumulate at the injury site in the KO mice, as shown by immunostaining for CD45 (Fig. 2B).

Fig. 2.

Increased inflammation during vascular lesion formation in A2bAR KO mice. (A) The levels of proinflammatory cytokines in the plasma of WT and A2bAR KO mice at day 1 and at 4 weeks after femoral injury. The cytokine basal level was measured in the plasma of normal WT and A2bAR KO mice. The data shown represent averages ± SD (n = 3). The way the sham injury is typically carried out in this model is by a sham in one femoral artery (e.g., left leg) and injury on the other femoral artery (right femur) in the same mouse undergoing surgery. This provides a control sample under the same physiological conditions but precludes measurements of plasma levels in sham vs. injury. With this approach, at day 1 after injury the increased cytokine levels should be due to both vascular injury and the surgical procedure. However, at 4 weeks after injury the major source of proinflammatory cytokines is from vascular injury, rather than from the surgical procedure. (B) Leukocyte immunohistochemistry staining with anti-CD45 or IgG (as control) in femoral arteries at 1 week after guidewire-induced injury. (Magnification: ×600.) Red arrows point to DAB substrate brown staining indicative of positive signal. Vascular lumen is indicated by “L.” As expected, no significant number of leukocytes was detected at 4 weeks after injury (data not shown).

Elevated CXCR4 Accompanies Neointima Formation After Injury in A2bAR KO Mice.

In our search for mechanisms that could account for the observation that A2bAR KO mice display greater vascular lesion formation after injury, we considered studies reporting the important role of the chemokine receptor CXCR4 in cell migration and neointimal hyperplasia after arterial injury (12, 13). Interestingly, CXCR4 is up-regulated in the A2bAR KO lesions (Fig. 3A and SI Fig. 8 A and B). It is abundant in the thrombus, in accordance with reported high levels of this receptor in platelets (14). To determine the expression of CXCR4 in cells that typically contribute to thrombus and lesion formation, we examined the levels of CXCR4 protein by Western blotting in freshly derived platelets, macrophages, and leukocyte fractions. Most platelet components, except for the nucleus and the transcription machinery, are transferred from megakaryocytes to platelets during platelet biogenesis. Therefore, megakaryocytes are used to study the regulation of genes that encode proteins found in platelets. CXCR4 expression is up-regulated in all of these cell types derived from A2bAR KO mice as compared with WT mice (Fig. 3B). CXCR4 levels tend to be higher in aortic smooth muscle cells prepared from KO mice as compared with those prepared from WT mice, but not with statistical significance (n = 3; data not shown). This could be because of phenotypic changes resulting from passaging (typically two) of the primary cells used here. To further verify CXCR4 elevation on the cell surface of A2bAR KO cells, flow cytometry was used to study the expression of CXCR4 in leukocytes, and its level is ≈2-fold greater in the KO cells as compared with control (SI Fig. 9). It should be pointed out that TNF-α, which is elevated in the plasma of A2bAR KO mice (Fig. 2A), has previously been reported to up-regulate the expression of CXCR4 (15).

Fig. 3.

Up-regulated CXCR4 expression in A2bAR KO mice. (A) CXCR4 in femoral arteries of WT and A2bAR KO mice at 1 week after femoral artery injury. No staining is noted with control IgG. At this early collection time, thrombus formation is typically noted, as also shown here in the lumen. Staining is mainly noted in the thrombus, and some is also in the vascular layer of the KO mice (a higher-magnification image of ×1,000 is shown in SI Fig. 8A). A representative image is shown. (Magnification: ×600.) Vascular lumen is indicated by “L.” The staining intensity was quantitated by calculating mean staining index by using the Image-Pro Plus software (Media Cybernetics), showing an average increase of 3-fold in CXCR4 staining in the A2bAR KO injured femoral artery area compared with WT (based on measurement of seven fields randomly taken throughout the stained sections; see SI Fig. 8B). (B) CXCR4 protein expression (43 kDa) in platelets, megakaryocytes, macrophages, and leukocytes as measured by Western blot analysis. β-Actin expression is used as loading control in each cell type. Quantification was performed by using NIH ImageJ software (version 1.62; http://rsb.info.nih.gov/ij). The data shown represent the averages ± SD of three experiments using a two-tailed paired Student t test. *, P < 0.05 (considered statistically significant).

The A2bAR Gene Promoter Activity Is Elevated in Proliferating VSMC, and Their Proliferation Is Up-Regulated in A2bAR KO Cells.

The proliferation potential of primary VSMC derived from A2bAR KO mice was also evaluated. Our A2bAR KO mouse model contains the prokaryotic gene for β-gal inserted under the control of the A2bAR gene promoter instead of the deleted gene (10). A2bAR gene activity, as reflected by β-gal assay, is significantly noted in VSMC of the injured vessel compared with the noninjured KO vessel (SI Fig. 10 A and B). Because A2bAR activation and cAMP elevation typically inhibit VSMC proliferation (4, 16–18), it is possible that during injury the gene is activated, thereby suppressing uncontrolled VSMC proliferation. In accordance with this contention, aortic smooth muscle cells isolated from KO mice proliferate in response to mitogens without the lag typical of cultured WT primary mouse VSMC (SI Fig. 11). Consistent with this, the cell proliferation markers proliferating cell nuclear antigen (PCNA) and cyclin D1 (19–21) are up-regulated in KO cells as compared with control (Fig. 4). The augmentation in proliferation potential of A2bAR KO cells, although moderate, could contribute to the increase in lesion formation.

Fig. 4.

Expression of the cell cycle markers PCNA and cyclin D1 in primary VSMC derived from WT and A2bAR KO mice. Proteins were derived from WT or A2bAR KO aortic smooth muscle cells at days 3, 5, and 7 in culture and subjected to Western blotting as detailed in Materials and Methods, using β-actin as a loading control. Data shown are representative of three experiments for days 3 and 7 and of two experiments for day 3.

Bone Marrow A2bARs Contribute to Neointima Formation After Injury.

Macrophages are a major source of plasma TNF-α. In a previous study we showed that the bone marrow cells are mostly responsible for augmented levels of TNF-α in A2bAR KO mice (10). Taking into account the elevation of this cytokine in the injured KO mice (Fig. 2A) and its pro-proliferative effect on VSMC (22), we sought to determine the contribution of bone marrow cells to vascular response to injury in A2bAR KO mice. To this end, A2bAR KO mice were irradiated and transplanted with WT bone marrow cells and vice versa. As shown in Fig. 5 and in SI Fig. 12 and SI Table 2, the majority of the lesion in A2bAR KO mice could be recapitulated in irradiated WT mice implanted with A2bAR KO bone marrow cells. Of note, in the chimeric mice the intima to media ratio (I/M) tends to be greater than in the nonchimeric corresponding WT or A2bAR KO mice (Fig. 1 and Table 1); however, these differences are not statistically significant.

Fig. 5.

Bone marrow cell-derived signals contribute to vascular lesion formation in A2bAR KO mice. Quantitation is shown as intima/media (I/M) ratios of femoral arteries at 4 weeks after injury from WT→WT (WT bone marrow transplanted into WT mice), KO→WT (KO bone marrow transplanted into WT mice), KO→KO (KO bone marrow transplanted into KO mice), and WT→KO (WT bone marrow transplanted into KO mice) transplanted groups. Data are expressed as average I/M ratio ± SEM for each group (n = 4 mice per group) using a two-tailed unpaired Student t test. Differences of P ≤ 0.05 were considered statistically significant.

Discussion

Vascular injury represents a critical initiating event in the pathogenesis of various vascular diseases, including atherosclerosis, restenosis, graft vasculopathy, organ transplantation, sepsis, etc. (23). Various models of vascular injury have been introduced, including denudation of the endothelium by mechanical injury (wire, balloon catheters) (24, 25). Luminal mechanical injury leads to endothelial denudation and platelet activation, which contribute to inflammatory cell recruitment. The presence of activated inflammatory cells at the site of the vascular injury leads to the release of vasoactive molecules, cytokines, and growth factors, which can induce the migration and proliferation of VSMC. Ultimately this contributes to hyperplasia. The traditional hypothesis for VSMC hyperplasia is that these dedifferentiated cells migrate from the media to the subendothelial space, where they proliferate and contribute to neointima formation after injury to the endothelium (23). Accumulating evidence from human and animal models suggests that not only medial VSMC but also progenitor cells contribute to neointima formation after vascular injury (12, 13, 26–29).

In the current study we show that guidewire-induced vascular lesion in A2bAR KO mice could be recapitulated in irradiated WT mice implanted with A2bAR KO bone marrow cells. Hence, bone marrow cells' A2bARs significantly contribute to tissue protection from vascular injury. In addition to bone marrow cell contribution, the increased ability of A2bAR KO VSMC to proliferate might be an important, although clearly not the only, regulator of neointima formation. After vascular injury, leukocytes and progenitor cells are attracted to injured tissues by adhesion molecules and chemokines (30). CXC chemokine stromal cell-derived factor 1 promotes stem cell mobilization and bone marrow engraftment, as well as vascularization during embryogenesis (31). CXCR4 is the only G protein-coupled seven-transmembrane receptor of stromal cell-derived factor 1. CXCR4 is expressed on the surfaces of multiple cell types, such as macrophages, lymphocytes, hematopoietic stem cells, bone marrow stromal cells, megakaryocytes, and platelets (32–36). TNF-α is a major regulator of CXCR4 in several cell types (15, 37, 38). CXCR4 is involved in the trafficking of hematopoietic progenitor and stem cells in embryologic hematopoiesis, in vasculogenesis, and in cardiogenesis (13, 39). Increasing evidence suggests that vascular progenitor cells participate in vascular repair and remodeling after injury (40, 41) and that this process is regulated by the stromal cell-derived factor 1/CXCR4 signal axis (12, 29, 42). CXCR4 also controls migration of inflammatory cells (43), and its activation mediates platelet aggregation (44), which is important for thrombus formation. As shown here, CXCR4 is up-regulated in injured vessels of A2bAR KO mice and in various cells that typically contribute to lesion formation. This could be because of TNF-α-mediated regulation and/or other mechanisms, such as direct or indirect signaling via the A2bAR, which are yet to be explored. Our earlier transplantation experiments indicated that bone marrow cells are the major source of elevated TNF-α in A2bAR KO mice (10). TNF-α is elevated in the injured A2bAR KO mice, while, as mentioned above, there is evidence to suggest that TNF-α up-regulates CXCR4 (15). Intriguingly, the protective effect of the A2bAR on vascular injury mirrors the one described for TNF-α-null mice (45). A very recent report confirmed our previously described increase in TNF-α levels in A2bAR KO mice at baseline and upon stimulation with lipopolysaccharide (10). In addition, based on experiments involving application of an A2-type adenosine receptor ligand, 5′-N-ethylcarboxamidoadenosine and A2bAR antagonists in control and A2bAR KO cells and mice, it was concluded that this receptor can also exert influences via interaction with signaling pathways unrelated to A2bAR activation (46). Future studies are needed to examine the direct effect of adenosine analogs and/or TNF-α on the control of CXCR4 expression in WT versus A2bAR KO cells.

In summary, our study highlights the significance of the A2bAR in controlling CXCR4 expression in vivo and in protecting against vascular injury via bone marrow-derived signals.

Materials and Methods

Animals.

A2BAR KO/β-gal knockin mice (C57BL/6 × 129S background) were originally generated in our laboratory (10). In the current study the mice used were on a pure C57BL/6 background strain (after breeding), confirmed by the PCR-based gene marker analysis MAX-BAX (Charles River Laboratories). In all experiments, WT and KO mice were strain-, sex-, and age-matched (10–12 weeks old unless otherwise indicated). All procedures were performed according to the Guidelines for Care and Use of Laboratory Animals published by the National Institutes of Health. Throughout this study, all animals received humane care that was in agreement with the guidelines of and approved by the Institutional Animal Care and Use Committee of the Boston University School of Medicine.

Mouse Femoral Artery Injury Model.

Ten-week-old male WT and A2bAR KO mice or 16-week-old transplanted mice were subjected to guidewire-induced endothelial denudation of the mouse femoral artery followed by analysis essentially as described in refs. 24, 47, and 48 and as detailed in SI Text.

Bone Marrow Transplantation.

Ten-week-old WT or A2bAR KO male mice were irradiated with a total dose of 12.5 Gy from a ¹³⁷Cs source. On the day of irradiation, bone marrow cells were harvested from 10-week-old WT and from A2bAR KO female donor mouse femurs. The bone marrow cells were subjected to red blood cell lysis as described previously (10) and injected through the tail vein into the irradiated mice at a dose of 2 × 10⁶ cells per recipient in 0.3 ml of phosphate buffered saline (PBS). Transplantation efficiency was confirmed by Jaridld and Jaridlc PCR as in ref. 10 using blood DNA isolated 5 weeks after transplantation and by bone marrow β-gal staining as previously described (10). The mice were subjected to femoral artery injury 6 weeks after transplantation. Tissue and blood samples were collected 4 weeks after injury unless otherwise indicated. Histopathological and immunohistochemical examinations were performed on the femoral arteries as in ref. 48.

Analysis of β-Gal Expression in Femoral Artery.

Mice were anesthetized with isoflurane and perfused through the left heart ventricle with 20 ml of PBS (pH 7.4) at a rate of 4 ml/min. Perfusion with fixative [30 ml of freshly made 2% paraformaldehyde in PBS (pH 7.4)] was proceeded for 15 min at 2 ml/min, followed by perfusion with PBS for 10 min. Various tissues were dissected out and stored in ice-cold PBS before staining for β-gal activity as described previously (10) and in SI Text.

Immunohistochemistry.

Paraffin sections of mouse femoral artery were deparaffinized and rehydrated, followed by high-temperature antigen retrieval [by placing sections in 10 mM citric acid (pH 6) in a 700-W microwave for 2 min for three cycles with 2-min intervals between each cycle], and then cooled to room temperature for 20 min. The sections were blocked with 10% normal goat serum (Vector Laboratories) for 1 h at room temperature and then incubated with primary antibody overnight at 4°C. In some cases sections were blocked with 0.3% hydrogen peroxide before blocking with serum. Staining was revealed by using goat anti-biotinylated secondary antibody (Vector Laboratories) at a dilution of 1:200 for 30 min at room temperature. After washing with PBS, sections were incubated with ABC-AP reagent or ABC reagent (Vector Laboratories) for 30 min at room temperature. Vector Red alkaline phosphatase substrate or DAB substrate (Vector Laboratories) was used to develop the red or brown positive signal by incubation for 5–20 min. Mouse monoclonal antibody staining was pursued by using a M.O.M. kit (Vector Laboratories) according to the manufacturer's protocol. The primary antibodies used were á-actin (mouse monoclonal at a dilution of 1:1,000; Sigma), CD45 (rat anti-mouse CD45 monoclonal at a dilution of 1:25; BD Pharmingen), and CXCR4 (rat anti-mouse monoclonal at a dilution of 1:20; R & D Systems).

Isolation and Culture of Mouse Megakaryocytes, Platelets, Macrophages, Leukocytes, and Aortic VSMC.

Bone marrow cells were isolated as described before (49). After 5 days in culture in IMDM (Invitrogen) supplemented with 10% FBS and thrombopoietin (50), a megakaryocyte-rich fraction was harvested as in ref. 50. Macrophages were isolated from peritoneal cavities of mice as described in ref. 10. Mouse aortic smooth muscle cells were isolated, cultured, and counted as described in ref. 10 and in SI Text. Leukocytes and platelets were isolated as follows: 500–600 μl of blood was collected by heart puncture with a syringe containing 100 μl of Aster–Jandi anticoagulant (85 mM Na-citrate/69 mM citric acid/20 g/liter glucose, pH 4.6). The blood was centrifuged at 600 × g for 8 min at room temperature. The plasma was removed and centrifuged at room temperature for 3 min at 400 × g. The pellet containing the leukocyte-rich fraction was collected, and the supernatant plasma was spun down at 16,000 × g for 5 min at room temperature. The platelets in the pellet were washed once with PBS at room temperature for 5 min at 16,000 × g.

Western Blot Analysis.

Whole cell pellets were collected and subjected to Western blotting as in ref. 10 and in SI Text. Membranes were probed with a 1:1,000 dilution of mouse monoclonal cyclin D1 antibody (Santa Cruz Biotechnology), a 1:1,000 dilution of mouse monoclonal PCNA antibody (Calbiochem), or a 1:5,000 dilution of CXCR4 rabbit polyclonal antibody (Abcam) overnight at 4°C. The membranes were probed with the following secondary antibodies from Santa Cruz Biotechnology: goat-anti-mouse or goat-anti-rabbit IgG at a 1:10,000 to 1:20,000 dilution for 1 h at room temperature. The protein bands were visualized by incubation with Millipore Immobilon Western Chemiluminescent HRP Substrate (Millipore). The same membranes were stripped (2% SDS/100 mM 2-mercaptoethanol/62.5 mM Tris·HCl) and reblotted with a 1:10,000 dilution of mouse monoclonal β-actin-specific antibody (Sigma) to confirm equal loading.

Measurements of Cytokines in Plasma.

Whole blood was collected from each mouse at 1 day and at 4 weeks after femoral artery injury through heart puncture. Blood was collected into BD Vacutainer spray-coated K2 EDTA tubes (BD Diagnostic Systems), and plasma was isolated from anticoagulated blood after centrifugation at 800 × g. Commercial ELISA kits were used to measure mouse TNF-α and IL-6 (ELISA Ready-SET-Go!; eBioscience) according to the manufacturer's instructions.