Endothelial Cells Modulate the Proliferation of Mural Cell Precursors via Platelet-Derived Growth Factor-BB and Heterotypic Cell Contact
Abstract—Embryological data suggest that endothelial cells (ECs) direct the recruitment and differentiation of mural cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal cells (10T1/2 cells) and induce their differentiation toward a smooth muscle cell/pericyte lineage. The present study was undertaken to investigate cell proliferation in these cocultures. ECs and 10T1/2 cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-β had no effect on EC-stimulated 10T1/2 cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 cells in the absence of contact. The cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated cell sorter analysis revealed that ECs and 10T1/2 cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 cell cocultures inhibited the growth of both cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-β in coculture-induced mural cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-β. Furthermore, the decreased proliferation of 10T1/2 cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-β in the 10T1/2 cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal cells and that heterotypic cell-cell contact and soluble factors play a role in growth control during vessel assembly.
Blood vessels are relatively simple structures composed of 3 cell types. Endothelial cells (ECs) form the inner luminal lining, mural cells (smooth muscle cells or pericytes) constitute the medial layer, and fibroblasts make up the outer adventitial layer. The process of embryonic vessel formation, termed vasculogenesis, begins with the differentiation of angioblasts into ECs, which subsequently form tube-like structures.1 2 3 Studies of vascular development reveal that cells of the vessel wall are added at later stages of vessel assembly.4 5 This developmental sequence suggests that the ECs are responsible for the acquisition of mural cell layers.
The nature of the cell-cell interactions that occur during vessel formation and their mediators is difficult to discern from in vivo studies. However, in vitro analysis suggests a role for the chemoattractant and mitogenic properties of several soluble factors known to be produced by ECs such as platelet-derived growth factor (PDGF),6 basic fibroblast growth factor (bFGF),7 and heparin-binding epidermal growth factor (HB-EGF).8 Evidence is particularly strong for the involvement of PDGF. Studies of developing vessels in the placenta reveal that PDGF receptor-ligand distribution is consistent with the recruitment of mesenchymal cells by the endothelium.9 ECs of developing vessels produce PDGF-BB, whereas PDGF receptor (R)β is expressed by developing smooth muscle cells (SMCs) and the surrounding mesenchyme. Furthermore, mice deficient for PDGF-B10 11 or PDGF-Rβ12 exhibit abnormal vessel development. The importance of the autocrine and paracrine roles of vascular growth factors is further emphasized in mice deficient for vascular endothelial growth factor (VEGF)13 and transforming growth factor (TGF-β),14 which also display defects in vessel formation.
To examine EC-mesenchymal interactions more directly, we have established an in vitro system in which we can study the migration, proliferation, and differentiation of vascular cells, processes that occur during the formation of the vasculature. This system allows us to examine the contributions of cell-cell contact, as well as autocrine and/or paracrine factors that have been implicated in vessel development. Using this model system, we demonstrated previously that ECs can direct the migration of multipotent mesenchymal cells (10T1/2 cells) in a process mediated by PDGF-B.15 Furthermore, once ECs and 10T1/2 cells make contact, 10T1/2 cells are induced to differentiate toward an SMC lineage, a phenotypic change that is mediated at least, in part, by TGF-β. The present study was undertaken to determine the effect of coculture on cell proliferation and the relative role of diffusible factors and cell-cell contact.
We found that in noncontacting cocultures, the proliferation of 10T1/2 mesenchymal cells was significantly increased in the presence of ECs, in a paracrine response mediated by PDGF-B. In cocultures, in which the 10T1/2 mesenchymal cells contacted the ECs, the growth of both cell types was suppressed. Conditioned media from contacting cocultures also inhibited growth of both ECs and 10T1/2 cells. We believe that information gained from these studies will aid in understanding the regulation of vessel development and the pathogenesis of certain vascular diseases.
Materials and Methods
ECs were isolated from bovine aortas by collagenase digestion as previously described.16 The cells were grown on uncoated tissue culture plastic in DMEM with 10% calf serum (CS) supplemented with glutamine, penicillin, and streptomycin and were used through passage 20. 10T1/2 cells (American Type Culture Collection, No. CCL 226) were grown in DMEM with 10% FCS and 4.5 g/L glucose. All cells were maintained at 37°C in a humidified incubator. DMEM with 2% CS was used for all experiments unless otherwise specified.
Cocultures of bovine aortic endothelium (BAE) cells and 10T1/2 cells were established in one of two models. (1) One assay is a modification of an underagarose assay system described by Nelson et al17 in which 2 cell types are cocultured in 5-mm wells created 2 mm apart in 1% agarose/1% BSA in DMEM. 10T1/2 cells were plated in one well, and BAE cells, a control vehicle, or growth factors were added to the other well. Cells were incubated for 2 days at 37°C and then treated as described below for bromodeoxyuridine (BrdU) labeling. During the 2-day coculture, the cells migrate but do not make contact. (2) In the second system, referred to as a direct coculture, equal numbers of both cell types were plated simultaneously and incubated for up to 7 days.
BAE cells or 10T1/2 cells were plated in uncoated 24-well plates at 1×104 cells per well in 2% CS/DMEM. Twenty-four hours later, the cells were challenged with the growth factor of interest, including PDGF-BB, PDGF-AB, PDGF-AA, VEGF, bFGF, or TGF-β1 at the specified concentrations. Conditioned media (CM) from BAE cells, 10T1/2 cells, and cocultures were also assayed for their effect on cell proliferation at a final concentration of 50% CM in DMEM/2% CS. Antibodies that neutralize the activity of TGF-β1, TGF-β2, and TGF-β3 (10 μg/mL; Genzyme, Inc) were included in some CM experiments. Cell number of unchallenged cells was determined 24 hours after plating to assess plating efficiency. After 72 hours, cells were rinsed with PBS, trypsinized, and counted using a Coulter counter (Coulter Corporation). All proliferation assays were conducted in quadruplicate, and each set of experiments was performed at least 3 times.
To determine the labeling index of the cells, BAE cells and 10T1/2 cells were plated in the underagarose assay, as described above, and allowed to incubate for 48 hours. In some experiments, neutralizing antibodies against PDGF-A and PDGF-B (1:200; Genzyme, Inc), bFGF (1:200; generously provided by Dr Michael Klagsbrun, Harvard Medical School), or TGF-β1, TGF-β2, and TGF-β3 (10 μg/mL; Genzyme, Inc) were incorporated directly into the agarose. BrdU labeling was achieved by addition of 10 μmol/L BrdU for 1 hour at 37°C. Cells were then fixed in 4% paraformaldehyde and stained with the use of a mouse monoclonal antibody to BrdU (6 μg/mL), according to the manufacturer’s instructions (Boehringer-Mannheim). Labeling indices were determined by counting the number of labeled cells out of a total of 100 cells at the front or back of each well.
Analysis of Cell Proliferation in Contacting Cocultures
The effect of contact on cell proliferation in the EC-10T1/2 cell cocultures was determined by prelabeling both cell populations with different “permanent” fluorescent dyes and coculturing the cells for 7 days. The cells were trypsinized, the total cell number determined by Coulter counting, and the proportion of each cell type determined by fluorescence-activated cell sorting (FACS). BAE cells were prelabeled with Cell Tracker Blue (Molecular Probes, Inc), a fluorescent blue dye retained by the cells for up to 20 doublings. For labeling, BAE cells were rinsed with PBS and incubated for 60 minutes at 37°C in DMEM/10% CS containing 10 μmol/L Cell Tracker Blue. After the dye was removed, the cells were rinsed with fresh medium and incubated in DMEM/10% CS overnight at 37°C before use.
10T1/2 cells were prelabeled with PKH26 (Sigma), a red fluorescent dye, which is retained for up to 100 doublings.18 Cells were trypsinized to a single cell suspension, pelleted, rinsed with PBS, and the number determined using a Coulter counter. The 10T1/2 cells were then resuspended at 2×106 cells per 100 μL of Diluent C (provided with the PKH26 dye). An equal volume of 40 μmol/L PKH26 dye was added, followed by incubation for 2 to 3 minutes with gentle agitation. The reaction was stopped by adding 2 volumes of FCS and 7 mL of 0.1% BSA in PBS. Cells were layered onto 3 mL FCS and pelleted. After centrifugation, cells were rinsed with DMEM/10% FCS, resuspended in fresh medium, and incubated overnight at 37°C.
Labeled cells were plated in DMEM/2% CS, either alone at 3×105 cells/100-mm dish or in coculture (1:1) at the same density. Cocultures and solo cultures were incubated for 7 days at 37°C; the cells were then trypsinized, pelleted, and resuspended at approximately 1×106 cells/mL in PBS. The cells were sorted on the basis of their different fluorescent profiles using an Epics Elite flow cytometer (Coulter Electronics). The effect of coculture on proliferation was determined by comparing the number of BAE cells and 10T1/2 cells after coculture to the cell numbers in the solo cultures.
Solo cultures of BAE cells or 10T1/2 cells were prepared by plating 1.5×105 cells in a 100-mm dish. For the coculture, 1.5×105 of each cell type were plated together in a 100-mm dish. After 7 days, the cells were harvested for RNA with 1 mL/plate of RNAzol B (Tel-Test).
Total cellular RNA (10 μg per sample) was separated by electrophoresis through a 1% agarose gel containing 2.2 mol/L formaldehyde in gel running buffer (0.2 mol/L MOPS, 50 mmol/L sodium acetate, and 10 mmol/L EDTA). RNA was transferred onto a GeneScreen Plus nylon membrane (NEN Research Products) with 10× SSPE (1× SSPE=150 mmol/L NaCl, 10 mmol/L NaH2PO4 · H2O, and 1 mmol/L EDTA). The membrane was rinsed vigorously in 2× SSPE and baked 2 hours at 80°C to immobilize RNA. Prehybridization was carried out for >3 hours at 42°C in 10 mL of hybridization solution (50% deionized formamide [Amresco], 5× SSPE, 5× Denhardt’s solution [5 Prime → 3 Prime], 100 μg/mL denatured salmon sperm DNA [5 Prime → 3 Prime], and 1.0% SDS [BioRad]). cDNA probes (human PDGF-B and murine PDGF-Rβ) were labeled using Ready-To-Go DNA labeling beads (Pharmacia Biotech) and were purified by centrifugation through MicroSpin S-200 HR columns (Pharmacia Biotech). Labeled probes were typically added to the hybridization mix at a concentration of 1.5×106 counts per minute (cpm)/mL of hybridization solution. Hybridization was carried out for >18 hours at 42°C.
Membranes were typically washed for 20 minutes at room temperature in 2× SSPE, 0.1% SDS, 20 minutes at room temperature in 1× SSPE, 0.1% SDS, and 15 minutes at 50°C in 0.5× SSPE, 0.1% SDS. The hybridized membrane was used to expose X-OMAT AR film (Eastman Kodak Co). Before reprobing, the blot was stripped for >1 hour in 0.1× SSPE, 0.25% SDS that had been heated to 100°C.
We have previously established in vitro coculture systems using ECs and 10T1/2 mesenchymal cells to model various aspects of blood vessel assembly. Using the underagarose assay system, we have shown that ECs direct the migration of 10T1/2 cells, in a process mediated by PDGF-B.15 19 In the present study, we were interested in determining whether coculture influences cell proliferation and, if so, whether soluble factors and/or contact are involved.
Cell Proliferation in EC-10T1/2 Cocultures in the Absence of Contact
10T1/2 cells were induced to migrate toward the ECs in coculture. To determine the effect of coculture on cell proliferation, 10T1/2 cells were cultured with BAE cells in the underagarose assay (without contact) and then pulsed with BrdU. Darkly labeled nuclei of cells, which had incorporated BrdU, were evident in 10T1/2 cells cultured alone (not shown) as well as in 10T1/2 cells cocultured with BAE cells, although there were clearly more labeled cells in the cocultures (Figure 1a⇓). The labeling indices of 10T1/2 cells, grown under both conditions, were determined. For 10T1/2 cells cultured alone (in DMEM/2% CS), the labeling indices were equivalent among the cells on both sides of the well; 22% among the cells on the side of the well closest to the other well and 23% among the cells on the side furthest from the other well (Figure 2a⇓). When 10T1/2 cells were cocultured with BAE cells, the labeling index of the 10T1/2 cells closest to the BAE cells increased to 52%, whereas the labeling index of the cells at the back of the well, farthest from the BAE cells, remained at 22%.
Effects of EC-Derived Soluble Factors on 10T1/2 Cell Proliferation
To determine which EC-derived soluble factor(s) might be responsible for the increased 10T1/2 cell proliferation, we first directly challenged 10T1/2 cells with various soluble effectors, including the 3 PDGF isoforms, bFGF, VEGF, and TGF-β1. PDGF-BB increased 10T1/2 cell proliferation in a dose-dependent manner with a maximal effect at 5 ng/mL. bFGF induced a modest increase in 10T1/2 cell growth (Figure 3b⇓), whereas PDGF-AB and PDGF-AA had no significant effect (Figure 3a⇓); neither VEGF (Figure 3c⇓) nor TGF-β1 (Figure 3d⇓) altered 10T1/2 cell proliferation.
The possible role of these factors in the EC-stimulated 10T1/2 cell proliferation was assessed by incorporating specific neutralizing antisera in agarose of the underagarose coculture assay. Blocking the action of PDGF-B completely prevented the increase in BrdU labeling observed in the presence of BAE cells (Figure 1b⇑), reducing the labeling index of the 10T1/2 cells closest to the BAE cells from 52% to 24% (Figure 2a⇑). The specificity of this effect was demonstrated by the fact that neutralization of PDGF-A (Figure 2b⇑), bFGF (Figure 2c⇑), or TGF-β (data not shown) had no effect on EC-induced 10T1/2 cell labeling index. The labeling index of the BAE cells in 10T1/2-BAE cocultures was approximately 40%, equivalent among cells at the front and back of the well, and was not influenced by the inclusion of neutralizing antisera against PDGF-A, PDGF-B, or TGF-β (data not shown).
Cell Proliferation in Contacting Cocultures of ECs and 10T1/2 Cells
In our previous studies, we found that contact between ECs and 10T1/2 cells induced differentiation of 10T1/2 cells toward a mural cell lineage.15 We therefore aimed to determine if cell proliferation was also modulated as a result of this intercellular interaction. To assess the effects of BAE-10T1/2 cell contact on cell proliferation, BAE cells and 10T1/2 cells were prelabeled with Cell Tracker Blue and PKH26, respectively, and were cocultured for up to 7 days. Total cell number was determined by electronic counting and the proportion of each cell type was determined by FACS. When BAE cells and 10T1/2 cells were grown in direct coculture, 10T1/2 cell growth and BAE cell growth were inhibited 47% and 43%, respectively, relative to the cells cultured alone (Figure 4a⇓ and 4b⇓).
To determine if this inhibition was the result of heterotypic contact only or if a soluble factor was generated on contact, media conditioned by the EC-10T1/2 cell cocultures were tested for their effect on the proliferation of each cell type. CM collected from solo cultures were also examined. Media conditioned for 3 days by subconfluent BAE cells or for 7 days by confluent BAE cells and assayed at a final concentration of 50% stimulated a 3- to 4-fold increase in BAE and 10T1/2 cell number compared with controls (Figure 5a⇓ and 5b⇓). Media collected from solo cultures of 10T1/2 had no effect on either cell type. The addition of media conditioned for 3 days by BAE-10T1/2 cell cocultures inhibited the proliferation of 10T1/2 cells and BAE cells by 61% and 48%, respectively, compared with cells cultured alone.
We had previously observed that contact between ECs and 10T1/2 cells leads to the activation of TGF-β.15 Because TGF-β is a known inhibitor of EC growth,20 21 we suspected that TGF-β might be responsible for the growth inhibition observed in the cocultures. Thus, antibodies that neutralize TGF-β1, TGF-β2, and TGF-β3 were used to determine if TGF-β plays a role in the growth inhibition observed in the BAE-10T1/2 cell cocultures and/or in the inhibitory effects of the coculture CM. We first performed a control experiment to demonstrate the effectiveness of the antisera. ECs were treated with 1 ng/mL TGF-β1 in the presence or absence of the neutralizing antisera (10 μg/mL). As expected, treatment with 1 ng/mL TGF-β1 significantly reduced (42%) EC number from 30 963±763 to 20 197±789 after 3 days. The presence of the neutralizing antisera against TGF-β1, TGF-β2, and TGF-β3 completely inhibited the EC growth-suppressing effects of TGF-β1 (29 700±3720). Presence of the antisera, however, did not influence the inhibitory effect of the cocultures (Figure 4a⇑ and 4b⇑) or have a reproducible effect on the growth inhibition caused by the CM (Figure 6a⇓ and 6b⇓). Similar results were obtained when soluble TGF-β receptor II was used to block TGF-β activity in the direct cocultures (data not shown).
We also investigated the possibility that the growth inhibition observed in the contacting cocultures might be due to the downregulation of PDGF-B in ECs or PDGF-Rβ on the 10T1/2 cells. However, we found that coculture had no effect on the expression of PDGF-B mRNA in the ECs (Figure 7⇓, top) or PDGF-Rβ mRNA in the 10T1/2 mesenchymal cells (Figure 7⇓, middle). The slight decrease in PDGF-Rβ mRNA seen at 7 days was not a consistent finding (data not shown).
Assembly of mature blood vessels requires coordinated homotypic and heterotypic cellular interactions between and among ECs, mural cells, and their mesenchymal precursors. The formation of mature vessels reflects a balance among paracrine and autocrine growth factors and involves vascular cell migration, proliferation, and differentiation. We have developed a coculture system that recreates some of the individual steps of vessel assembly. Using this system, we have previously shown that ECs can recruit undifferentiated mesenchymal cells and induce their differentiation toward an SMC/pericyte fate.15
The present study focuses on the understanding of the modulation of cell proliferation during various stages of vessel assembly and the role of diffusible factors and intercellular contact. We show that the presence of ECs stimulates the proliferation of 10T1/2 cells, a multipotent mesenchymal cell line, and that this stimulation is specifically mediated by PDGF-B. Furthermore, we demonstrate that the growth of both ECs and mesenchymal cells is inhibited on heterotypic contact. The observed growth inhibition is not associated with reduced expression of PDGF-B in ECs or PDGF-Rβ downregulation in 10T1/2 cells in the contacting cocultures. Furthermore, although TGF-β is known to influence the differentiation of mesenchymal cells under the same culture conditions,15 the growth suppression does not appear to be mediated by TGF-β.
Our coculture results provide strong evidence for a role of PDGF-B as a paracrine effector in EC-directed mesenchymal recruitment and proliferation. Proliferating ECs have been shown to synthesize PDGF both in vitro and in vivo.22 Because PDGF has a short half-life in the circulation in vivo (<2 minutes),23 it is therefore thought to operate locally. Our observation that PDGF mediates the EC effects on 10T1/2 cell migration and proliferation is consistent with other reports of mitogenic and chemotactic PDGF-BB effects on a variety of mesenchymally derived cells, including fibroblasts24 and SMCs.25
Consistent with our in vitro observations, ECs of developing vessels have been reported to express PDGF-B mRNA and protein, whereas the surrounding undifferentiated mesenchymal cells have been shown to express the PDGF-Rβ.9 Mouse embryos deficient in PDGF-B have defects in cardiac development, dilated large vessels, and malformed kidneys.11 Interestingly, the kidney glomeruli lack mesangial cells, a cell type considered to be in the SMC/pericyte lineage.26 Mice deficient for PDGF-Rβ show similar defects in glomerular development.12 More recent data indicate that certain microvasculature beds of PDGF-B null mice lack pericytes.10 The authors hypothesized that this phenotype resulted from a lack of mesenchymal cell migration and/or proliferation, an interpretation corroborated by our results. This is further supported by analysis of PDGF-Rβ wild-type/mutant chimeric embryos in which there is an 8-fold reduction in aortic SMCs and pericytes.27
The fact that some cell types that express the PDGF-Rβ are normal in the Rβ-deficient mice is most likely due to compensation by PDGF-Rα. The PDGF-Rα is also implicated in vessel formation; in Patch mice, which are deficient in the PDGF-Rα, cardiovascular defects are characterized by reduced numbers of SMCs.28 Similarly, PDGF-A null embryos develop emphysema due to a deficiency of alveolar myofibroblasts.29 In other systems, PDGF-A has been shown to play a role in gastrulation, mediating the migration of PDGF-Rα–expressing mesodermal cells across the PDGF-A–producing ectoderm.30 31 Hence, virtually all of the phenotypes observed in the PDGF ligand-deficient mice and receptor-deficient mice can be explained by absence of appropriate cell-cell signaling governing migration and proliferation.
During blood vessel assembly, the EC-recruited mesenchymal cells come to associate with ECs, making contacts that are retained in mature, adult vessels and are thought to be necessary for maintenance of the quiescent state.32 Consistent with this concept, we have shown that contact between ECs and 10T1/2 cells leads to the differentiation of mesenchymal cells toward a SMC/pericyte fate.15 To determine if this heterotypic intercellular contact also affects the cell proliferation, ECs and 10T1/2 cells were prelabeled with different fluorescent dyes, grown in direct contact, and then separated by FACS. We found that the growth of both BAE cells and 10T1/2 cells was inhibited. The fact that this inhibition took place when the cells were still subconfluent indicates that the observed growth inhibition was not density-induced. Of note, coculture had no effect on the expression of PDGF by ECs or PDGF-Rβ in 10T1/2 cells, indicating that the reduced proliferation was not secondary to a decrease in EC-produced PDGF or decreased ability of the 10T1/2 cells to respond. Furthermore, media conditioned by growth-inhibited cocultures suppressed the growth of solo cultures of BAE cells and 10T1/2 cells, indicating that growth inhibition was mediated, at least in part, via a soluble effector(s).
We knew from our previous studies that coculture of ECs with 10T1/2 cells, SMCs, or pericytes leads to the activation of TGF-β.15 21 TGF-β has not only been shown to induce mesenchymal cell differentiation15 but also to suppress EC proliferation21 and migration.33 However, the neutralization of TGF-β in the EC-10T1/2 cell cocultures, or their CM, had no significant effect on the growth inhibition of either 10T1/2 cells or ECs. We, therefore, suspect the presence of another, yet unidentified, inhibitor. Previous studies from our laboratory have demonstrated that postconfluent ECs produce a SMC inhibitor34 ; CM from sparse, proliferating ECs stimulated SMC proliferation, whereas CM from postconfluent ECs suppressed SMC growth. The growth inhibitor in the EC CM was not attributable to TGF-β or other known SMC inhibitors, including heparin or prostaglandins. CM from postconfluent ECs also inhibited the growth of 10T1/2 cells. Thus, it is possible that the same EC-derived effector mediates the growth suppression observed in the EC-10T1/2 cocultures.
Vessel assembly and maturation require the coordination of many seemingly divergent processes including migration, proliferation, and differentiation. Data obtained from our coculture system strongly implicate PDGF-B in the proliferation and migration of mesenchymal cells and TGF-β in the induction of SMC/pericyte differentiation. Furthermore, our observations indicate that heterotypic contact between ECs and mesenchymal cells plays an important role during vessel development. This study demonstrates the importance of paracrine interactions in development and adds to our understanding of the complexity of vessel formation and maturation.
This work was supported by EY05318 to P.A.D. S.A.R. was supported by the Harvard-Longwood Research Training Program Grant in Vascular Surgery (HL07734-02); K.K.H. was supported by an NIH Postdoctoral Fellowship (HL09037); and L.H.B. is supported by an NIH Medical Scientist Training Program Grant (GM07753). We gratefully acknowledge John Daley (Dana Farber Cancer Institute, Boston, Mass) for his assistance with the FACS analyses. We also thank Dr Diane Darland for her critical reading of this manuscript.
↵1 Both authors contributed equally to this study.
- Received August 10, 1998.
- Accepted October 29, 1998.
- © 1999 American Heart Association, Inc.
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