doi: 10.1161/hc5001.100663
(Circulation. 2001;104:3109.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Disordered Cellular Migration and Angiogenesis in cd39-Null Mice
From the Departments of Medicine (C.G., J.S., K.E., T.H., E.C., S.R.) and Pathology (C.S.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass.
*The first 2 authors contributed equally to this work.
Correspondence to Simon C. Robson, MD, PhD, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215. E-mail srobson{at}caregroup.harvard.edu
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Abstract |
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Background— Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 is the major ectonucleotidase of endothelial cells and monocytes and catalyzes phosphohydrolysis of extracellular nucleoside diphosphates (NDP) and triphosphates (NTP, eg, ATP and UTP). Deletion of cd39 causes perturbations in the hydrolysis of NTP and NDP in the vasculature. Activation of P2 receptors appears to influence endothelial cell chemotactic and mitogenic responses in vitro. Therefore, aberrant regulation of nucleotide P2 receptors may influence angiogenesis in cd39-null mice.
Methods and Results— In control mice, implanted Matrigel plugs containing growth factors were rapidly populated by monocyte/macrophages, endothelial cells, and pericytes, with the development of new vessels over days. In cd39-null mice, migrating cells were completely confined to the tissue-Matrigel interface in a clearly stratified manner. Absolute failure of new vessel ingrowth was consistently observed in the mutant mice. Linked to these findings, chemotaxis of cd39-null monocyte/macrophages to nucleotides was impaired in vitro. This abnormality was associated with desensitization of nucleotide receptor P2Y-mediated signaling pathways.
Conclusions— Our findings demonstrate a role for NTPDase1 and phosphohydrolysis of extracellular nucleotides in the regulation of the cellular infiltration and new vessel growth in a model of angiogenesis.
Key Words: adenosine • vessels • platelets • angiogenesis • endothelium
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Introduction |
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Angiogenesis is a highly complex multistep phenomenon that incorporates both formation of new capillaries and expansion from preexisting blood vessels.1,2 An associated increased permeability to plasma solutes results in the deposition of a provisional matrix in which fibrin is a major component.3,4 New vessel growth may be modulated by monocyte/macrophages that secrete angiogenic factors and metalloproteases that facilitate endothelial cell migration.5 Supporting cells are also essential for new vessel growth and angiogenesis, for example, smooth muscle cells in vascular maturation and arteriogenesis and pericytes in the protection of newly developing endothelial cell-lined tubes from rupture and regression.2,6
The in vivo Matrigel assay has previously been used to evaluate angiogenesis.7–9 This assay has certain advantages over other in vivo models, such as wound healing and tumor formation. The evaluation of reconstituted basement membrane matrices incorporating peptide growth hormones and other factors has facilitated evaluation of endothelial cell requirements for formation of capillary networks in vivo. Specifically, platelet derived sphingosine 1-phosphate (SPP) appears to be an important regulator of angiogenesis.10,11
Nucleotides bind to P2 receptors, of which the P2Y group initiate G protein-coupled signaling pathways.12 In this context, UTP has been shown to be mitogenic and chemotactic for endothelial cells in vitro.13 Interestingly, binding of angiostatin, a proteolytic fragment of plasminogen and potent antagonist of angiogenesis, to ATP synthase expressed on endothelial cells has been shown to mediate antiangiogenic effects.14
Uncertainty as to the mechanisms of such interactions prompted us to evaluate the role of nucleotides in angiogenesis using the cd39-null mouse model, in which aberrant regulation of nucleotide P2 receptors has been observed.15 CD39 (or nucleoside triphosphate diphosphohydrolase-1, NTPDase1) is the major vascular endothelial membrane NTPDase and hydrolyses nucleoside triphosphates and diphosphates, ultimately to the nucleoside analogues16; these products also have mitogenic effects on endothelial cells in vitro.17 The purpose of this study was to investigate new vessel growth in the Matrigel model of angiogenesis in cd39-null mice.
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Methods |
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Antibodies and Other Reagents
The polyclonal antibody anti-rat NG2 recognizes murine chondroitin sulfate proteoglycan, expressed on activated pericytes18; the macrophage marker used was rat F4/80 from Serotec. The monoclonal antibodies directed at mouse heparan sulfate proteoglycan (perlecan) and anti-laminin-ß2 chain were purchased from Chemicon or Pharmingen. The fluorescein-labeled monoclonal antibody anti-smooth muscle
-actin (clone 1A4) was used as a marker for pericytes and smooth muscle cells.19 Biotinylated monoclonal antibody anti-mouse CD31 recognizing platelet and endothelial cell adhesion molecule (PECAM)-1 anti-ß3 integrin subunit and anti-mouse CD144 recognizing VE cadherin were purchased from Pharmingen. The polyclonal antibody rabbit anti-mouse platelet-derived growth factor (PDGF)-ß receptor (Clone 958) was purchased from Santa Cruz Biotechnology. Polyclonal antibodies to vascular endothelial growth factor (VEGF) receptor-2 were from R. Brekken and P. Thorpe (University of Texas Southwestern Medical Center, Dallas). Biotinylated rabbit anti-mouse (Fab')2 and biotinylated pig anti-rabbit (Fab')2 were purchased from Dako. Biotinylated rabbit anti-rat IgG, fluorescein-conjugated goat anti-rabbit IgG, rhodamine-conjugated rabbit anti-rat IgG, and Texas Red avidin D were from Vector Laboratories. Normal rabbit, mouse, swine, and goat serum and nonimmune rat, rabbit, and mouse IgG were purchased from Sigma. The terminal deoxynucleotidyl transferase apoptosis kit and the proliferating cell nuclear antigen staining kit were purchased from R&D systems and Zymed and used according to the manufacturers’ instructions.
In Vivo Angiogenesis Assay
Mutant mice deficient in cd39 on the C57BL/6x129 svj strain were generated, validated, and characterized as we have described previously15,20; age- and sex-matched wild-type animals (C57BL/6x129 svj strain) were from Taconic. The animal experimentation protocol was reviewed and approved by the Animal Care and Use Committees of the Beth Israel Deaconess Medical Center. Mice were injected with 200 µL of Matrigel (Costar, Fischer Scientific) at a final concentration of 9.9 mg/mL, containing 1.4 µg/mL VEGF, 8 µg/mL fibroblast growth factor (FGF)-2, 116 µg/mL BSA (fatty acid-free) purchased from Sigma, and 500 mmol/L SPP from Biomol.10
Animals were euthanized at 7, 14, and 21 days. Matrigel-injected and control tissues were embedded, snap-frozen in isopentane, and stored at -70°C before sectioning with immunohistochemical staining.21
Immunohistology, Double Immunofluorescence Staining, and Confocal Microscopy
Immunohistology, double immunofluorescence staining, and confocal microscopy were performed exactly as previously described.21
NTPDase Activity and FACS Analysis
Monocyte/macrophages were harvested from the peritoneal cavity of wild-type and cd39-null mice after injection of 10 mL of PBS. Membrane-bound NTPDase activity was determined by measuring the amount of liberated inorganic phosphate (Pi) hydrolyzed from exogenous ATP (Sigma)22,23; 5 mmol/L tetramisole was added to inhibit alkaline phosphatase.
After isolation, naive peritoneal macrophages were also stimulated for 30 minutes (or for 24 hours) in RPMI+5% FCS, both at 37°C, with ATP 200 µmol/L, or with ATP
S 200 µmol/L, and then washed with PBS. Nonspecific binding of isospecific IgG was controlled by incubation with isospecific IgG conjugated to FITC and phycoerythrin, respectively. Cells were double-stained with phycoerythrin-conjugated rat anti-mouse CD11b in conjunction with FITC-labeled anti-mouse intercellular adhesion molecule (ICAM)-1 (or very late antigen-1, -4), respectively. Fluorescence-activated cell sorter (FACS) analysis was performed (FACScan, Becton Dickinson). Data were analyzed with CellQuest computer software (Becton Dickinson).
Transmigration Assays
Transmigration assays through an endothelial cell monolayer were performed.24 Murine 2F2B endothelial cells (ATCC) were plated on collagen type I-coated polycarbonate inserts with 3-µm pores (Costar, Fischer Scientific) and then incubated in RPMI containing 5% FCS overnight to attain full confluence. Wild-type or cd39-null peritoneal macrophages were added to the upper chamber and incubated at 37°C in 5% CO2 for 10 hours. In the lower chamber, culture media with chemoattractant factors at various combinations were added: ATP 200 µmol/L, serotonin 20 µmol/L, and monocyte chemoattractant factor-1 (MCP-1) 50 ng/mL. Nonadherent cells in both supernatant fluids were counted. In parallel experiments, inserts were precoated with collagen or Matrigel alone. Wild-type and cd39-null macrophages were plated on the upper surface of coated inserts. Cells were allowed to adhere and migrate over a 6-hour period. Cells on the lower surface were fixed in 2% ethanol and stained with 0.2% crystal violet (Sigma). Four fields of vision (x400) per well were randomly chosen, and cell counts were performed manually by 2 observers in a blinded fashion.
Statistical Analyses
Student’s t test (2-tailed) was used for the comparative analyses.
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Results |
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Angiogenesis Model
Four Matrigel plugs containing all 3 growth factors/additives from cd39-null mice and 4 matched Matrigel samples from wild-type mice were harvested at day 7. Six Matrigels containing all 3 additives from cd39-null mice and 6 Matrigel plugs from wild-type mice were harvested at day 14. Four Matrigel plugs containing all 3 additives from cd39-null mice and 4 Matrigels from wild-type mice were harvested at day 21. Two wild-type or mutant mice for each time point were injected with Matrigel plugs containing only VEGF/SPP and were also analyzed in parallel.
Tissues were analyzed at the 14-day point with regard to differences in alterations in vascular density at the interface between Matrigel and underlying tissue, as well as observed ingrowth of vessels into the matrix (n=6).
Wild-type mice displayed an increased vessel density at the interface as well as demonstrating substantial ingrowth of vessels into the Matrigel itself (Figure 1, a and c). Native vessels in the adjacent normal skin displayed signs of activation, indicated by the development of thin-walled, pericyte-poor vessels, called "mother vessels," with staining for CD31 (Figure 1, a and c). Newly formed vessels to a large extent were surrounded by NG2 and PDGF-ß receptor-expressing pericytes and were invested in a basement membrane, indicated by the presence of both perlecan and laminin (not shown). NG2 and PDGF-ß receptor-expressing pericytes, as well as monocyte/macrophages, were present at the leading edges of these connective-tissue septa. This pattern was followed by ingrowth of CD31-expressing endothelium (Figure 1, a and c; Table).
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In contrast, cd39-null mice did not develop full angiogenic responses at the interface or within the Matrigel itself (Figure 1, b and d). Vessels adjacent to the smooth muscle layer of the normal tissue showed some initial angiogenic responses, as indicated by the development of mother-vessel formation. Decreases in the expression of basement membrane components as well as increases in expression of the activation markers, ie, KDR, PDGF-ß receptors, NG2, and vß3 were noted in the cd39-null mice, albeit to a lesser degree than observed in the wild-type mice (Table). Migration of endothelium toward the interface and into the Matrigel was absent in the cd39-null mice (Figure 1).
In addition, pericyte and macrophage infiltration was absent in the null mice (Figure 2). An interface between the dermis and the Matrigel in wild-type mice was associated with connective tissue septa penetrating into the Matrigel and high cellularity within the Matrigel itself (Figure 2a); several of the unlabeled cells are considered fibroblasts. A compact cellular rim constituting the inflammatory zone at the interface between the dermis and the Matrigel, as well as the lack of cells within the Matrigel itself, was observed in cd39-null mice (Figure 2, c and e). Marked F4/80-positive macrophage infiltration into the Matrigel itself in wild-type mice contrasted with a compacted rim of F4/80-positive macrophages in the dermis at the dermis/Matrigel interface in cd39-null mice (Figure 2, c and e versus d and f).
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On closer examination, null monocytes, endothelial cells, and pericytes were present in distinct monocellular sandwich-type layers that approximated the interface between the normal tissue and the Matrigel (Figure 3).
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NTPDase Activity
The ATPase (ADPase) activity of the wild-type macrophages approximated 2090±200 (1780±110) Pi nmol · min-1 · well-1, whereas cd39-null macrophage activity was measured at 90±10 (30±10) Pi nmol · min-1 · well-1 (n=6; P<0.05).
Cytofluorometric Analyses
Median levels of wild-type macrophage cell surface coexpression of mß2 (MAC-1) and ICAM-1 increased significantly (n=6; P<0.05) after stimulation with 200 µmol/L ATP or ATPS (30 minutes; n=6). In contrast, mß2 and ICAM-1 coexpression levels in quiescent cd39-null macrophages were largely unaltered after either ATP or ATPS stimulation, respectively; an increase was not statistically significant compared with the basal levels (Figure 4).
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Transmigration of Monocyte/Macrophages Through Endothelial Cell Barriers, Collagen, and Matrigel Matrices
Spontaneous migration of wild-type monocyte/macrophages across the endothelial cell line monolayer approximated 4900±400 cells. Control monocyte/macrophages also migrated toward extracellular ATP (200 µmol/L; 22 300±3300). In response to MCP-1 (50 ng/mL), greater numbers of wild-type monocyte/macrophages migrated to the lower chamber across the monolayer (61 800±23 400; n=6). Mutant cd39-null monocyte/macrophages showed comparable levels of spontaneous migration at 6200±1200. cd39-null macrophages, however, migrated poorly toward a source of extracellular ATP (200 µmol/L; 8750±4100 P<0.05; n=4). The migratory response of null cells to MCP-1 (35 200±7300) was less than wild-type cellular migration responses (n=6, P<0.05).
Transmigration of wild-type and mutant macrophages was then tested on Matrigel-coated Transwell membranes (and collagen; not shown), in the absence of endothelial cells. As before, wild-type macrophages had low baseline migration levels; serotonin stimulation alone (20 µmol/L) did not substantially change the number of migrating macrophages (normalized to 120±20%), but ATP stimulation resulted in an increase of transmigrated cells (230±20%; P<0.05 and n=6 [Figure 5]). Costimulation with ATP and serotonin substantially boosted migration of wild-type cells to levels of 280±20%. MCP-1 alone increased migration to 450±20%, whereas MCP-1 in combination with ATP effected levels of 480±20%; n=6 (Figure 5). The number of nonstimulated cd39-null macrophages that spontaneously migrated was 80±30.0% of wild-type cells. Serotonin alone had no substantial effect on migration of cd39-null macrophages (98±20.0%).
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In contrast to wild-type macrophages, ATP stimulation did not significantly increase the transmigration of cd39-null macrophages (107±24%; n=6, not significant). ATP in combination with serotonin, however, significantly boosted migration of cd39-null macrophages to 219±19% (P<0.05; n=6). MCP-1 alone and MCP-1 in combination with ATP showed lesser effects on the transmigration of cd39-null macrophages (303±17% and 282±22%, respectively) and relative to wild-type controls (Figure 5).
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Discussion |
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The cd39-null mice have been shown to develop increased vascular permeability with tissue fibrin sequestration,15 which might have resulted in heightened angiogenic responses in vivo.25,26 However, we demonstrated almost total failure of the angiogenic response within the Matrigels containing SPP, FGF-2, and VEGF in vivo (Figures 1 and 2). Interestingly, there was complete stratification of the mutant cellular infiltrate at the Matrigel-tissue interface and an ordered distribution into the monocyte/macrophage, endothelial cell, and pericyte layers. This pattern suggested a stereotyped cellular migration into the Matrigel and emphasized the interdependence of these cells in the orchestration of the angiogenic responses in vivo (Figure 3).2 This observation is not unique to this model tested, because we have recently also shown disordered angiogenic responses to murine tumors when the cells were injected into the cd39-null mice (T.H., unpublished data)
Primary monocyte/macrophage abnormalities with dysfunctional recruitment, activation, and/or migration into the Matrigel might provide an explanation for the lack of angiogenesis seen in cd39-null mice. The deletion of cd39 removed the major NTPDase activity at the cell surface of monocyte/macrophages. In keeping with published data,27 extracellular ATP upregulated mß2 expression on control monocyte/macrophages; in contrast, ATP failed to induce surface expression of mß2 on macrophages from cd39-null mice (Figure 4). We then demonstrated that ATP had potent chemoattractive potential for wild-type monocyte/macrophages in vitro. Importantly, the migratory potential of cd39-null macrophages in response to ATP (or MCP-1) was substantially decreased compared with control monocyte/macrophages (Figure 5A). Moreover, in contrast to the wild-type cells, ATP failed to attract cd39-null macrophages and promote migration through the Matrigel in vitro (Figure 5).
Abnormalities in monocyte/macrophage regulation of integrins and chemotactic responses (Figures 4 and 5) suggested that G protein-coupled P2Y-receptor desensitization responses may have occurred after deletion of cd39, as previously observed for platelet P2Y1.15 Therefore, we examined the effects of serotonin, in combination with ATP, to bypass certain P2Y-mediated pathways, as previously validated for platelet aggregation.15 Serotonin acts via unique G protein-coupled receptors on macrophages.28 Costimulation with serotonin and ATP rapidly restored the migratory responsiveness of cd39-null macrophages through the Matrigel (Figure 5). The exact mechanisms for this remain under evaluation, but P2Y-sequestration and phosphorylation reactions have been demonstrated to be associated with this phenomenon.29
A further explanation for the failure of angiogenesis would be a primary inability of microvessels to react to appropriate angiogenic stimuli. Against this possibility is the observation that initial early events in angiogenesis were demonstrated in mutant mice, ie, mother-vessel formation,30 degradation of the basal lamina, and decreased expression of VE-cadherin31 (Figures 1 and 2; Table). Endothelium in cd39-null mice expressed CD31 (Figure 1) and the vß3 integrin (Table) that mediates adhesion and migration with respect to several components of the provisional matrix.32,33 Further analysis of functional activity of vascular endothelial integrins will be evaluated in the future.
Exogenous growth factors are implicated in the progressive neovascularization of Matrigel plugs in vivo. The importance of FGF-2 in this phenomenon is emphasized by the observation that Matrigel containing only VEGF and SPP failed to induce migration of blood vessels into the Matrigel (C.S., unpublished data). FGF-2 is thought to exert its proangiogenic effects either directly on endothelium or through an indirect effect via activation of monocytes/supporting cells.2,9
Our findings demonstrate that the phosphohydrolysis of extracellular nucleotides is important for both regulation of the cellular infiltrate at the initiation of angiogenesis and the progression of neovascularization. These observations may be pertinent to the understanding of new vessel growth in such human disease processes as cancer, rheumatoid arthritis, and diabetic retinopathy.
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Acknowledgments |
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These experiments were supported by NIH grants HL-57307 and HL-63972 (Dr Robson) and CA-50453 (to Dr H. Dvorak); also American Heart Association grant GIA 9650490N and grants from the German National Research Foundation to Dr Goepfert, from the Swedish Cancer Society, Konung Gustaf V:s 80-årsfond to Dr Sundberg, and from the Canadian Institute of Health Research to Dr Sévigny. We thank Dr H. Dvorak for his support of Dr Sundberg and encouragement for this work.
Received April 25, 2001; revision received October 11, 2001; accepted October 12, 2001.
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