CD73/Ecto-5′-Nucleotidase Protects Against Vascular Inflammation and Neointima Formation
Background— Although CD73/ecto-5′-nucleotidase has been implicated in maintaining vasoprotection, its role in regulating endothelial adhesion molecule or inflammatory monocyte recruitment (eg, in the context of vascular injury) remains to be defined.
Methods and Results— Compared with wild-type mice, CD73-deficient (CD73−/−) mice exhibit increased luminal staining and protein and transcript expression for vascular cell adhesion molecule (VCAM)-1 in carotid arteries. In vitro, aortic endothelial cells (ECs) from CD73−/− mice display an upregulation of mRNA and protein expression of VCAM-1, associated with increased nuclear factor (NF)-κB activity, as determined by chromatin cross-linking and immunoprecipitation or quantitative p65 binding assays. CD73−/− ECs and carotid arteries perfused ex vivo supported increased monocyte arrest under flow conditions, which was mediated by α4β1 integrin. After wire injury of carotid arteries, CD73 expression and activity were upregulated in wild-type mice, whereas neointimal plaque formation and macrophage content were increased in CD73−/− mice versus wild-type mice, concomitant with elevated NF-κB activation, luminal VCAM-1 expression, and soluble VCAM-1 concentrations. In contrast, reconstitution of wild-type mice with CD73−/− versus CD73+/+ BM did not significantly exacerbate neointima formation. Treatment with the specific A2A receptor agonist ATL-146e reversed the increased VCAM-1 transcript and protein expression in CD73−/− ECs and inhibited monocyte arrest on CD73−/− ECs. Continuous infusion of ATL-146e prevented neointima formation in CD73−/− mice.
Conclusions— Our data epitomize the importance of vascular CD73 in limiting endothelial activation and monocyte recruitment via generation of adenosine acting through the A2A receptor, providing a molecular basis for therapeutic protection against vascular inflammation and neointimal hyperplasia.
Received October 13, 2005; revision received February 28, 2006; accepted March 2, 2006.
The cell membrane–anchored surface enzyme CD73/ecto-5′-nucleotidase catalyzes the extracellular conversion of 5′-AMP to the purine nucleoside adenosine, which controls many physiological and pathophysiological events.1–4 This process represents the last step in the extracellular adenine nucleotide breakdown after CD39/ATP-diphosphohydrolase–dependent conversion of ATP to ADP and AMP. In endothelial cells (ECs), adenosine has been shown to inhibit the release of cytokines and the expression of adhesion molecules.5,6 Moreover, stable adenosine analogues and an adenosine receptor agonist can reduce the expression of adhesion molecules and neointimal lesion formation in murine models of atherosclerosis.7,8 A strong activity of CD73 is predominantly associated with the vascular endothelium of large conduit vessels, such as the aorta and carotid and coronary arteries.9 Recently, the genetic deletion of CD73 associated with an impaired extracellular generation of adenosine demonstrated its importance in modulating vascular tone and barrier function and in limiting inflammatory and prothrombotic responses by attenuating leukocyte adhesion and platelet function.9,10 Atherosclerosis is widely considered to be an inflammatory disease involving the expression of adhesion molecules and proinflammatory cytokines, the recruitment of leukocytes, and the activation of prothrombotic pathways.11 Because these findings implied a constitutive effect of CD73 in maintaining antiinflammatory vasoprotection, we were prompted to study its role in endothelial vascular cell adhesion molecule (VCAM)-1 expression and monocyte recruitment, its regulation by arterial injury, and ultimately its contribution to neointimal plaque formation in the context of atherosclerosis.
Clinical Perspective p 2127
Mouse Model of Carotid Artery Injury
Male wild-type CD73+/+ or littermate CD73−/− mice (NMRI background)9 or wild-type mice repopulated with wild-type or CD73+/+ bone marrow (BM) were fed normal chow. For 1 week before and up to 4 weeks after arterial injury,12 mice were fed an atherogenic diet containing 21% fat (0.15% cholesterol, 19.5% casein). Transluminal carotid artery injury12 was induced by 4 rotational passes of a 0.36-mm guidewire. One day before injury, some mice were anesthetized and underwent implantation of a primed 28-day osmotic pump (model 2004, Alzet, Durect Corporation, Cupertino, Calif) subcutaneously placed via transverse midscapular incision for continuous treatment with ATL (0.004 μg/kg per minute, 1% dimethyl sulfoxide, provided by G.W. Sullivan and J. Linden, University of Virginia).8 One day before fixation, mice were intraperitoneally injected with 100 mg/kg of 5-bromo-2′-deoxyuridine (BrdU, Sigma, St Louis, Mo). Arteries were harvested by in situ perfusion fixation with 4% paraformaldehyde and embedded in paraffin. Donor BM was prepared from femurs and tibias from wild-type or CD73−/− mice,12 and cells in phosphate-buffered saline (2×106) were administered by intracardial puncture to CD73+/+ mice 24 hours after an ablative dose of whole-body irradiation (2×5.5 Gy). Animal experiments were approved by local authorities and complied with German animal protection law.
Morphometry, Immunohistochemistry, and Enzyme Histochemistry
Neointimal and medial areas were quantified in serial 5-μm sections of common carotid arteries within a standardized distance from the bifurcation (50 to 250 μm) and stained with Movat pentachrome.12 Areas within the external elastic lamina, internal elastic lamina (IEL) and lumen were determined by computer-assisted planimetry (Diskus software, Hilgers, Königswinter, Germany). Neointimal area was defined as IEL-lumen, medial area as external elastic lamina-IEL. Relative cell content in the neointima was determined in serial sections (3 to 6 per mouse, 50 μm apart) within 300 μm from the bifurcation. Immunohistochemistry was performed using isotype controls (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), antibodies (Abs) to Mac-2 (CL8942AP, Cedarlane Labs, Hornby, Ontario, Canada), α-smooth muscle actin (α-SMA, clone 1A4), CD3 (PC3/188A, Dako, Glostrup, Denmark), CD73 (BD Biosciences, San Diego, Calif), p50 (NLS, Santa Cruz Biotechnology, Inc) reacted with fluorescein isothiocyanate (FITC)-conjugated monoclonal Ab (mAb, Sigma), VCAM-1 (C-19), and ICAM-1 (M-19, Santa Cruz Biotechnology, Inc) detected by a biotin-conjugated secondary Ab visualized by Vectastain ABC-AP and Vector Red Substrate kit (Vector Laboratories, Burlingame, Calif). Neutrophils were detected by specific esterase staining (naphthol-AS-d-chloracetate, Sigma). BrdU was labeled according to the manufacturer’s protocol (Zymed Laboratories, San Francisco, Calif), mAb binding to thrombin (K-20, Santa Cruz Biotechnology, Inc) was visualized by an avidin/biotin peroxidase–linked detection system (Vector), and sections were counterstained with Mayer’s hemalum (Merck Biosciences, Bad Soden, Germany). Apoptotic nuclei were detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining (Roche, Indianapolis, Ind). BrdU+ and TUNEL+ cells in the neointima were counted relative to all 4′6-Diamidino-2-phenylindole (DAPI) positive cells. Images were recorded using a Leica (Leica Microsystems, Wetzlar, Germany) DMLB microscope. CD73 activity and expression were quantified as previously described9 in uninjured arteries and in arteries 4 weeks after injury (3 sections per mouse) using AnalySIS software (Soft-Imaging Systems, Münster, Germany).
Cell Isolation and Culture
Mouse aortic ECs were isolated and cultured as described previously.13 Briefly, aortas cut into circular rings were opened longitudinally and placed luminal side down onto collagen gels. After 10 days, aortic segments were removed, and collagen gels were digested with 0.3% collagenase H. ECs were grown in complete medium and used at passages 2 to 8. Immortalized murine ECs served as controls. Some ECs were treated with ATL-146e (10 μmol/L) for 6 hours. MonoMac6 cells were maintained as described previously.14 Medial smooth muscle cells (SMCs) were isolated from uninjured arteries by enzymatic digestion12 and grown in SMC-GM2 medium (PromoCell, Heidelberg, Germany). Proliferation was assessed by CyQUANT Proliferation Assay (Molecular Probes), and migration was determined by counting cells that have migrated after scratch injury.
Immunofluorescence and Flow Cytometry
Confluent ECs on glass slides were fixed in methanol, permeabilized with 0.5% Triton-X, and reacted with p65 mAb (C-20, Santa Cruz Biotechnology, Inc). SMCs were fixed in 4% paraformaldehyde, stained with mAbs to α-SMA, VE-cadherin (C-19, Santa Cruz Biotechnology, Inc), or isotype control, and visualized by FITC-conjugated Abs. For flow cytometry, ECs were reacted with saturating concentrations of CD73 and VCAM-1 mAbs (BD Biosciences) or isotype controls. ECs permeabilized with 0.1% Triton-X were stained with von Willebrand factor (vWF) Ab (F-3520, Sigma) or isotype control detected by FITC-conjugated Abs and analyzed in a FACSCalibur (BD Biosciences). Specific mean fluorescence intensity was determined by subtracting control staining.
ChIP and NF-κB Binding Assays
Chromatin cross-linking and immunoprecipitation (ChIP) assays were performed as described in a previous study15 with the use of Abs recognizing subunits of the nuclear factor (NF)-κB family (Santa Cruz Biotechnologies), cytochrome c (H-104, Santa Cruz), or acetyl-histone H3 (Upstate, Charlottesville, Va). The following polymerase chain reaction (PCR) primers were used: 5′-GGACTTGGCTGGCTGT-CAGGTTAAAC-3′ and 5′-AGTGTCTGGGAAAAGTGTTCAGC-CTC-3′. Nuclei were isolated by incubation in 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES containing 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride) on ice. Debris was removed by centrifugation, and total protein was measured by Bio-Rad Dc (detergent compatible) assay. DNA binding was analyzed with the use of an ELISA plate assay by incubating nuclear extracts on immobilized oligonucleotide containing the NF-κB consensus site GGGACTTTCC (TransAM, Active Motif, Rixensart, Belgium).
Ex Vivo Perfusion of Carotid Arteries After Wire Injury
Carotid arteries were isolated, transferred onto a microscope stage (saline immersion objective), and perfused with MonoMac6 cells (106/mL) labeled with calcein-AM (Molecular Probes, Leiden, the Netherlands) in 3-(N-morpholino)propanesulfonic acid–buffered physiological salt solution at 4 μL/min.9,12 Cells were left untreated or pretreated with anti-α4 mAb (10 μg/mL; clone HP2/1, Immunotech, Marseille, France). Adhesive interactions with the vessel wall were recorded by using stroboscopic epifluorescence illumination (Drelloscop 250, Drello, Mönchengladsbach, Germany), and firmly adherent cells were counted after 10 minutes and expressed as a percentage of untreated control.
Polymerase Chain Reactions
RNA was prepared with the use of the RNAquous-Micro isolation kit (Ambion) and RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed into cDNA by Mo-MLV (Invitrogen, Paisley, UK). PCR was performed with 20 ng cDNA using Taq DNA polymerase (Promega, Mannheim, Germany) and specific primers (aldolase: 5′-AG-CTGTCTGACATCGCTCACCG-3′, 5′-CACATACTGGCAGCGCTTCAAG-3′; CD739; vWF: 5′-GGAATTCTGCTCAGTGGGGTGGATG-3′, 5′-CGGATCCGGGCTCACGTCCA TGCGC-3′; and VCAM-1: 5′-AGAGAAACCATTTATTGTTGACATCTCC-3′, 5′-AGAGAAA CCATTTATTGTTGACATCTC-3′). Products were separated by agarose gel electrophoresis, and densitometry was performed (Scion-Image software, Frederick, Md). Real-time PCR was performed by use of the QuantiTect SYBR-Green PCR kit and specific primer pairs (VCAM-1: 5′-CACTCTGCCTCTGTTTGGGTTCA-3′, 5′-GAATTACTGAAGGGGGAGACTACAC-3′; GAPDH: 5′-CCA-CAGCCTTGGCAGC-3′, 5′-CACTCAAGATTGTCAGC-3′).
Laminar flow assays were performed as previously described.16 MonoMac6 cells pretreated with and without anti-α4 (10 μg/mL, Immunotech) were perfused at 1.5 dyne/cm2. The numbers of firmly adherent cells were quantified after 4 minutes in multiple fields, recorded by video microscopy, and expressed as cells/mm2.
Western Blotting and Immunoassay
After perfusion with phosphate-buffered saline, carotid arteries were excised and boiled in Laemmli buffer. Western blots for β-actin (clone AC-74, Sigma) and VCAM-1 (sc-1504, Santa Cruz) were performed by using the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK) and analyzed by densitometry (Scion-Image software). Soluble VCAM-1 (sVCAM-1) concentrations in mouse serum were quantified by sandwich ELISA following the manufacturer’s instructions (R&D Systems, Minneapolis, Minn).
Data represent mean±SEM and were compared by 2-tailed Student t test or 1-way analysis of variance followed by Newman-Keuls post hoc test where appropriate (InStat software, GraphPad, San Diego, Calif). Values of P<0.05 were considered to be statistically significant.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agreed to the manuscript as written.
Carotid arteries of CD73−/− but not wild-type mice perfused ex vivo have been found to support a substantial accumulation of monocytes on the endothelium.9 To elucidate underlying mechanisms, we investigated the expression of the endothelial adhesion molecule VCAM-1 in carotid arteries by immunofluorescence. Although VCAM-1 was hardly detectable in carotid arteries of wild-type mice, consistent with a marginal constitutive expression, staining for VCAM-1 was strongly increased along the endothelial lining of carotid arteries in CD73−/− mice (Figure 1A). In contrast, luminal ICAM-1 expression was detectable in wild-type arteries but decreased in CD73−/− arteries (not shown). Expression of adhesion molecules was further analyzed by reverse transcription (RT)-PCR in arteries collected from wild-type or CD73−/− mice. Although transcripts for CD73 could be detected only in arteries of wild-type but not CD73−/− mice (Figure 1B), confirming the genetic deletion of CD73 (Figure 1B), expression of VCAM-1 mRNA was low in wild-type arteries but markedly increased in CD73−/− arteries (Figure 1B), in line with the immunofluorescence results. In contrast, ICAM-1 mRNA expression was unaltered (not shown). These differences were substantiated by Western blotting of whole carotid lysates, revealing a marked upregulation of VCAM-1 protein in CD73−/− versus wild-type arteries (Figure 1C).
Because staining of VCAM-1 was mostly confined to the luminal lining, we further analyzed its expression in cultured aortic ECs isolated from wild-type or CD73−/− mice. Strong mRNA expression of vWF and distinct immunofluorescence staining for VE-cadherin but not α-SMA (Figure 2A and online Data Supplement) confirmed the endothelial phenotype of isolated wild-type and CD73−/− ECs. Although CD73 mRNA was detected only in wild-type but not CD73−/− ECs (Figure 2A), VCAM-1 mRNA expression was upregulated in CD73−/− versus wild-type ECs, consistent with results obtained in carotid arteries. Protein expression was analyzed by flow cytometry. Although both wild-type and CD73−/− ECs displayed positive staining for vWF, an absence of CD73 was confirmed in CD73−/− ECs (Figure 2B). Compared with wild-type ECs, the surface protein expression of VCAM-1 was increased in CD73−/− ECs (Figure 2B and 2C). Together, these results imply that the constitutive balance in the expression profile of arterial ECs is shifted toward a more proinflammatory phenotype in the absence of CD73.
Given that adenosine can block the activation of NF-κB in different cells17 and given the pivotal role of NF-κB in the transcriptional control of proinflammatory and antiapoptotic genes,18,19 including VCAM-1, we tested whether CD73 deficiency modulates the nuclear translocation of p65 as a marker for NF-κB activity by immunofluorescence in vitro. Compared with wild-type ECs, the proportion of cells with nuclear staining for p65 was significantly increased in CD73−/− ECs (19.4±2.0% versus 49.1±3.6%, respectively, P<0.0001; see arrows in Figure 2D). To substantiate differences in NF-κB activation, we performed ChIP analysis to assess NF-κB binding to specific motifs and transcriptional activity in the VCAM-1 promoter. Although no signals were obtained using a control Ab against cytochrome c, a pronounced increase in binding of NF-κB subunits and acetylated histone H3 to the VCAM-1 promoter was evident in CD73−/− compared with wild-type ECs (Figure 2E). In accordance, a quantitative p65 binding assay confirmed increased DNA binding of NF-κB in CD73−/− versus wild-type ECs (Figure 2F). These data indicate that CD73 deficiency is associated with high constitutive NF-κB activity.
Because very late antigen (VLA)-4 binding to VCAM-1 is known to mediate mononuclear cell adhesion,20 we explored the propensity of CD73−/− versus wild-type ECs to support monocyte adhesion under flow conditions and the contribution of VCAM-1 to monocyte recruitment in CD73−/− carotid arteries in situ. The arrest of monocytic cells, compared with wild-type ECs, was significantly increased on CD73−/− ECs (Figure 3A) and inhibited by Ab blockade of the VCAM-1 receptor VLA-4 (Figure 3A). Similarly, increased monocyte recruitment in carotid arteries of CD73−/− mice perfused ex vivo9 was significantly inhibited by blockade of VLA-4 (Figure 3B). Background arrest of monocytes in wild-type arteries was negligible and unaffected by blocking VLA-4 (not shown). Our data reveal the importance of VLA-4/VCAM-1 interactions for increased monocyte arrest in arteries of CD73−/− mice.
Because inflammatory leukocyte recruitment is instrumental in neointimal hyperplasia,21 lesions in carotid arteries were analyzed after wire-induced injury to investigate the role of CD73 in neointimal plaque formation. One week after injury, no differences in neointimal and medial areas were observed in wild-type versus CD73−/− carotid arteries (Figure 4A; medial areas are not shown). Adenosine can function as a potent antiinflammatory signal for neutrophils in vivo.22 Indeed, neutrophil accumulation tended to be augmented in CD73−/− versus wild-type arteries 1 week after injury, without reaching statistical significance (Figure 4B). Although the relative content of macrophages was significantly increased in CD73−/− versus wild-type arteries (Figure 4B), the relative content of CD3+ T cells was unaffected (not shown). The CD73-dependent adenosine formation at the endothelium-platelet interface has been shown to limit thrombotic events, with CD73−/− mice displaying enhanced thrombus formation.9 One week after injury, intense staining for thrombin was observed along the luminal lining of CD73−/− but not wild-type arteries (Figure 4C), whereas differences in cellular proliferation (as assessed by staining of BrdU+ cells) or apoptosis (as assessed by the number of TUNEL+ nuclei) were not observed (not shown; see online Data Supplement). Four weeks after injury, deficiency in CD73 significantly enhanced plaque formation, as evidenced by an increase in neointimal area compared with neointimal area in CD73+/+ mice; medial areas were unaltered (Figure 4D and 4E). Quantitative immunofluorescence revealed a significant increase in the relative content of macrophages and also SMCs in the neointima of CD73−/− mice (Figure 4F), whereas the content of CD3+ T cells was unchanged (not shown). The elevated SMC content could be related to a significant increase in the proliferation (129.1±11.5% of wild-type, P<0.05) but not migration (94.7±10.0%) of SMCs isolated from CD73−/− versus CD73+/+ mice. Immunofluorescence staining revealed that the upregulation of luminal VCAM-1 expression in CD73−/− versus wild-type carotid arteries was even more pronounced after wire injury than it was in uninjured CD73−/− arteries (Figure 4G; uninjured arteries are not shown) and that this expression was associated with increased nuclear translocation of p50 as a marker of NF-κB activity in luminal cells (Figure 4H). Notably, serum concentrations of sVCAM-1 were significantly increased in CD73−/− compared with wild-type mice 4 weeks after injury (Table). Notably, CD73−/− but not wild-type arteries frequently contained erythrocytes within neointimal lesions (0.9±0.3% versus 0.1±0.1% area, respectively, *P<0.05; see arrows in Figure 4D), suggesting that these may have been trapped within a thrombus.
Because CD73 is also expressed by circulating leukocytes,10 a BM transplantation model was used to dissect a contribution of CD73 on circulating cells to the injury response. Four weeks after injury, neointimal plaque formation was slightly but not significantly increased in CD73+/+ mice reconstituted with CD73−/− BM versus CD73+/+ BM (Figure 4E), implying that resident vascular ECs provide the prime source of local CD73 limiting neointimal hyperplasia. These data reveal that a proinflammatory EC phenotype in CD73−/− mice promotes neointimal hyperplasia and monocyte recruitment. Notably, both expression and enzymatic activity of CD73 were upregulated after arterial injury at the shoulder and along the luminal lining covering the neointimal plaques (Figure 5A and 5B). This implies that a compensatory upregulation of CD73 may occur after injury.
To investigate whether the loss of extracellular adenosine is responsible for the shift toward a more proinflammatory state in CD73−/− mice, the effects of a specific A2A adenosine receptor agonist (ATL-146e) were explored. In vitro, treatment with ATL-146e dose-dependently reduced the elevated VCAM-1 mRNA expression in CD73−/− ECs to levels seen in wild-type ECs as analyzed by PCR (Figure 6A and 6B). Flow cytometry further demonstrated that ATL-146e reversed the increased surface protein expression of VCAM-1 in CD73−/− ECs and slightly diminished its expression in wild-type ECs (Figure 6C). Concomitantly, the reduction in VCAM-1 expression by ATL-146e was paralleled by an inhibition of the monocyte arrest to CD73−/− ECs, whereas monocyte arrest to wild-type ECs was not affected by the treatment with ATL-146e (Figure 6D).
To functionally analyze underlying mechanisms of CD73 deficiency in vivo, wild-type and CD73−/− mice were treated by continuous infusion with ATL-146e for 4 weeks. Confirming previous findings,8 administration of ATL-146e significantly inhibited neointima formation 4 weeks after injury in wild-type carotid arteries compared with control wild-type arteries. Importantly, ATL-146e completely prevented neointimal hyperplasia in CD73−/− carotid arteries compared with control CD73−/− arteries (Figure 6E and 6F) and reduced the neointimal area to levels observed in wild-type mice. These data show that the more proinflammatory phenotype due to the absence of CD73 can be completely reversed by activation of the adenosine A2A receptor and imply that under physiological conditions CD73 generating extracellular adenosine may participate in the resolution of excessive inflammation.
In the present study, we show that luminal expression and mRNA of the endothelial adhesion molecule VCAM-1 is constitutively upregulated in carotid arteries of CD73−/− mice versus wild-type mice. Moreover, aortic ECs isolated from CD73−/− mice display higher VCAM-1 transcript and surface protein levels than do wild-type ECs. In contrast, ICAM-1 expression was decreased in arterial CD73−/− ECs but unaltered in whole artery samples. Moreover, the upregulation of luminal VCAM-1 in CD73−/− versus wild-type carotid arteries was even more pronounced after wire injury than in uninjured CD73−/− arteries. Notably, serum levels of sVCAM-1, postulated to correlate with inflammation and the extent of atherosclerosis,23 were significantly increased in CD73−/− versus wild-type mice 4 weeks after injury. Accordingly, adenosine (as the active metabolite generated by CD73) dose-dependently inhibits VCAM-1 but not ICAM-1 expression in activated human ECs in vitro,5 whereas the adenosine analogue 3-deazaadenosine or an adenosine receptor agonist inhibited both VCAM-1 and ICAM-1 expression in diet- or injury-induced arterial lesions in vivo, respectively.7,8 Beyond an antiinflammatory function of adenosine in activated endothelium, our data reveal a crucial role of CD73 in the constitutive regulation of endothelial adhesion molecules and inflammatory homeostasis. Differential effects of CD73 on ICAM-1 expression in ECs versus whole arteries may be attributable to its presence or effects in adjacent vascular cell types (eg, SMCs).
Adenosine binding to the A2A receptor can counteract the stimulation of VLA-4 expression and binding to VCAM-1 in neutrophils via a cAMP/protein kinase A–mediated pathway.24 Moreover, adenosine receptor activation on activated macrophages has been shown to inhibit the release of tumor necrosis factor-α, which can act directly on ECs to increase leukocyte adhesion.25 Thus, it is conceivable that under physiological conditions, adenosine catalyzed by CD73 and shed from ECs may sustain an antiinflammatory state in leukocytes circulating in the marginal pool, a mechanism absent in CD73−/− ECs. This is supported by findings that basal cAMP levels are diminished in platelets of CD73−/− mice, consistent with a constitutive stabilization of blood components by adenosine.9
In line with increased VCAM-1 expression, monocyte arrest was markedly increased in CD73−/− ECs in vitro and predominantly mediated by VLA-4/VCAM-1. Moreover, the in situ accumulation of monocytes in carotid arteries of CD73−/− mice perfused ex vivo9 was inhibited by blocking VLA-4, confirming a crucial role of its endothelial ligand VCAM-1 in mediating monocyte recruitment to CD73−/− carotid arteries. Although VLA-4 binding to VCAM-1 is known to mediate mononuclear cell adhesion to early atherosclerotic endothelium in carotid arteries in apolipoprotein E–deficient mice,20 our results identify VLA-4/VCAM-1 as the primary receptor-ligand pair crucial for proinflammatory monocyte recruitment in CD73−/− carotid arteries. Notably, this shift toward a proinflammatory phenotype in the absence of CD73 was prevented by treatment with ATL-146e, as evident by a reduction of VCAM-1 mRNA and protein expression in CD73−/− ECs. In addition, monocyte arrest to CD73−/− ECs was substantially inhibited after exposure to ATL-146e.
The molecular mechanisms by which CD73 prevents a proinflammatory endothelial phenotype remain to be solved. Previous data have demonstrated that adenosine can block NF-κB activation in different cell types.17 Given the pivotal role of NF-κB in the transcription of multiple proinflammatory and antiapoptotic genes,14,18,19 we sought to determine whether CD73 deficiency might modulate NF-κB activation. Indeed, NF-κB activity was increased in CD73−/− ECs in vitro, as evident by ChIP analysis of NF-κB and acetylated histone H3 binding to the VCAM-1 promoter and quantitative p65-DNA binding assays. This implies that the absence of CD73 metabolites (namely, adenosine), which would usually limit NF-κB activation, leads to constitutive NF-κB activity and transcriptional upregulation of VCAM-1.
Leukocyte infiltration mediated by sequential action of adhesion molecules is essential in the pathogenesis of atherosclerosis.11,26 Diminished activation of VCAM expression after treatment with the adenosine analogue 3-deazaadenosine or the adenosine A2A receptor agonist ATL-146e has been shown to reduce leukocyte recruitment and thus fatty streak and neointima formation induced by atherogenic diet or vascular ligation injury.7,8 Our data demonstrate that CD73 deficiency and the ensuing proinflammatory EC phenotype contributes to enhanced neointima formation in carotid arteries after wire injury. Whereas neutrophil accumulation tended to be higher in CD73−/− carotid arteries 1 week after injury, the neointimal content of macrophages was significantly enhanced at 1 week and further increased at 4 weeks after injury. Because neutrophils constitute the first line of inflammatory defense, the decline of neutrophils at 7 days might mask differences at earlier time points. With neointimal areas being equivalent at 1 week, the increase in macrophage content together with indifferent effects of CD73 deficiency on apoptosis and proliferation implies a dominant role of sustained monocyte recruitment in promoting neointima formation in CD73−/− arteries. An increase in luminal thrombin causing platelet activation may support a proinflammatory phenotype in CD73−/− arteries with deficient adenosine synthesis. Moreover, treatment with the adenosine receptor agonist ATL-146e attenuated neointima formation in wild-type mice and completely prevented neointimal plaque formation in CD73−/− mice. This indicates that adenosine synthesis and subsequent activation of A2A receptors in CD73+/+ mice constitutes an underlying mechanism by which CD73 protects against inflammation.
Given the expression of CD73 in leukocytes,10 circulating cells recruited to the site of injury may provide an important source of local CD73 in limiting the response to injury. The reconstitution of wild-type animals with CD73−/− BM compared with CD73+/+ BM, however, did not significantly exacerbate neointima formation. Polymorphonuclear leukocytes rapidly release 5′-AMP, requiring endothelial CD73 for effective conversion of 5′-AMP to adenosine, but they also generate adenosine within minutes of their activation. Moreover, extracellular endothelial adenosine receptor signaling was found to decrease leukocyte adhesion to ECs and paracellular permeability.10,27 Although our data do not preclude the possibility that circulating cells may provide an additional source of adenosine available at the endothelial surface,28 endogenous adenosine generated by resident CD73-expressing cells appears to be of prime importance in controlling neointimal hyperplasia. A protective role of CD73 in circulating cells could be unveiled by reconstituting CD73−/− mice with wild-type BM, which was not performed and may thus represent a limitation of the present study. Of note, CD73 expression and activity was increased 4 weeks after wire injury, inferring that a compensatory upregulation of CD73 participates in the physiological resolution of excessive inflammation during vascular remodeling by increasing local adenosine concentrations.
Interestingly, intralesional erythrocytes were frequently identified in neointimal lesions of CD73−/− but not wild-type arteries, in line with an increased overall susceptibility toward thrombotic events in CD73−/− mice.9 Inasmuch as recent findings indicate that erythrocytes within atherosclerotic plaques may represent an atherogenic stimulus associated with coronary plaque instability,28 intralesional erythrocytes may contribute to the formation of neointimal lesions in CD73−/− arteries.
Although extrinsic addition of adenosine or activation of its receptor can convey protection against atherosclerosis and inflammation, an interference with intrinsic physiological pathways involved in the adenosine metabolism has not been previously shown. Our data indicate that CD73 is crucially involved in the finely tuned constitutive regulation balancing proinflammatory and antiinflammatory mechanisms in the macrovasculature. These findings may inspire an approach elevating or mimicking CD73 activity or substituting its metabolites to reduce vascular inflammation and lesion formation. Currently, adenosine receptor agonists are gaining attention for use in tissue protection and vasoprotection.29 Because CD73 can be shed from vascular endothelium into the circulation while remaining functionally active (J. Schrader et al, unpublished data, 2005), the exogenous application of recombinant enzyme, which has been shown to reverse hypoxia-induced vascular leakage in CD73−/− mice,10 may be a feasible option to prevent cardiovascular events. In parallel, treatment with statins has been found to trigger activity of CD73, a mechanism that may contribute to the limitation of infarct size after coronary occlusion.30 Taken together, our data may help to provide a molecular basis for the therapeutic modulation of CD73, opening novel avenues for inhibiting vascular inflammation and neointimal hyperplasia.
This study was supported by Deutsche Forschungsgemeinschaft grants (WE 1913/5, SFB612-B6, and SFB542-B8/C12) and by the Interdisciplinary Center for Clinical Research BIOMAT.
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In large conduit vessels, such as the aorta and carotid and coronary arteries, the endothelial cell membrane–anchored surface enzyme CD73/ecto-5′-nucleotidase catalyzes the extracellular conversion of 5′-AMP to the purine nucleoside adenosine. The metabolism of adenosine has been shown to modulate vascular tone and barrier function as well as inflammatory and prothrombotic responses by altering leukocyte adhesion and platelet function. We show that CD73 is crucially involved in the finely tuned constitutive regulation, balancing proinflammatory and antiinflammatory mechanisms in the macrovasculature. Mice with a genetic deficiency of CD73 exhibited a constitutive upregulation in the activity of nuclear factor-κB, a proinflammatory transcription factor, and in the expression of vascular cell adhesion molecule-1, an endothelial adhesion molecule, and demonstrated an increase in the neointimal plaque formation and macrophage accumulation after wire injury of carotid arteries. Application of an adenosine receptor agonist indeed demonstrated that adenosine synthesis and subsequent activation of the A2A receptor constitutes the underlying mechanism by which CD73 protects against inflammation. Of note, CD73 expression and activity were increased after wire injury, inferring that a compensatory upregulation of CD73 may participate in the physiological resolution of inflammation during vascular remodeling. These findings provide a molecular basis for the therapeutic modulation of CD73 and further corroborate strategies of elevating CD73 activity and substituting or mimicking its metabolites to reduce vascular inflammation and lesion formation. Interestingly, treatment with statins has been found to trigger the activity of CD73, a mechanism that may limit infarct size after coronary occlusion but may also limit excessive vascular remodeling, and may thus represent an applicable therapeutic option.
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.595249/DC1.