Angiogenesis Research Center, Department of Medicine, Dartmouth-Hitchcock Medical Center, Dartmouth College, Lebanon, New Hampshire 03756
Submitted 19 February 2004 ; accepted in final form 11 June 2004
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ABSTRACT |
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metalloproteinase; plasminogen activator; p38 MAPK; thrombospondin 1; Sp1
The matrix proteins in the microenvironment are known to modulate cellular activities. On the basis of studies performed using fibronectin, collagen, matrigel, and fibrin matrices, it has been proposed that traction forces exerted by cells on viscoelastic substrata induce reorganization of the vicinal matrix into cords that provide positional information for the development of capillary-like structures (13, 26, 57, 58). However, endothelial cell morphogenesis into tubelike structures depends on random motility (50) and exposure to extracellular matrix proteins, as well as synthesis of endogenous proteins by the endothelial cells in response to extracellular stimuli (27, 39, 49).
Transforming growth factor (TGF)-1 is one of the numerous proteins that are critical to the assembly of endothelial and supportive cells into efficient blood vessels. After the binding of active TGF-
1 with its receptors, the vertebrate gene products related to mothers against decapentaplegia in Drosophila (SMAD) signaling proteins are activated and modulate the production of many extracellular matrix proteins involved in the regulation of angiogenesis, including plasminogen activator inhibitor (PAI)-1 and thrombospondin (TSP)1 proteins via p38 MAPK activation (52). TSP1 protein, in addition to activating the latent forms of TGF-
1 (47, 48), has been shown to inhibit lumen formation by microvascular endothelial cells (53). In support of the role of TSP1 in regulating TGF-
1 activity, studies of the phenotypes of TGF-
1 and TSP1 null mice reveal similar histopathological effects in multiple organ systems, particularly the lung and pancreas (12).
In vivo studies highlight the importance of TGF-1 signaling pathways in vasculogenesis and vessel wall integrity (42). Differentiation of extraembryonic endothelial cells into capillary-like tubules is defective in knockout mice that lack TGF-
1; the TGF-
receptors endoglin, T
R-II, activin receptor-like kinase (ALK)1, and ALK5; and the signaling intermediate protein SMAD5 (9). The vessel wall fragility observed in homozygous TGF-
1 and TGF-
receptor null mice is reminiscent of the vascular lesions in patients diagnosed with hereditary hemorrhagic telangiectasia (HHT). Mutations in endoglin or Alk-1 cause angiodysplastic lesions in distinct subsets of families diagnosed with HHT (2).
In addition to the significant knowledge gained from gene knockout and other studies, many details of the signal transduction pathways elicited by growth factors acting on endothelial cells are known. However, it is not clear how the various signaling pathways are integrated during the different phases of the angiogenic processes: endothelial cell shape changes, cellular alignment into a network of cords, lumen formation, and maturation into functional blood-transporting and nutrient- and gas-exchanging vessels. A better understanding of the molecular mechanisms underlying vascular patterning would greatly advance the effectiveness of strategies aimed at promoting or inhibiting new blood vessel formation. One potential application that could develop from an improved understanding of the signaling cues that determine the path of vessel growth would be directing new vessel growth to a particular tissue region of interest (e.g., an ischemic area or a wound) or interference with gene products that are critical in vascular patterning (e.g., cancer). This study describes capillary tube patterning in a fibrin matrix. The results suggest that TGF-1 treatment of endothelial cells causes a change in capillary tube patterning through a signaling cascade that is independent of p38 MAPK activation. The findings also suggest that the c-Jun NH2-terminal kinase (JNK) signaling pathway plays an important role in capillary tube formation in a fibrin matrix.
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MATERIALS AND METHODS |
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Preparation of BAEC aggregates. BAEC aggregates were prepared by a modification of a previously described method (44). BAEC monolayers were rinsed twice with PBS, detached using enzyme-free dissociation solution, centrifuged, and resuspended in serum-free DMEM. Cell suspensions were added to 96-well plates precoated with 1.5% agarose (50,00075,000 cells/well). The cells were incubated overnight at 37°C to allow formation of cell aggregates.
In vitro angiogenesis assay in fibrin gels.
Fibrin gels were prepared by addition of thrombin (1 U/ml) to a solution of plasminogen-depleted fibrinogen (3 mg/ml) in serum-free DMEM supplemented with 0.1% -aminocaproic acid and 2 µg/ml aprotinin. To make the underlying fibrin gel, 0.25 ml of fibrin solution was placed into each well of a 48-well culture plate and incubated at 37°C for 30 min. After the gels were polymerized, the endothelial cell aggregates were seeded onto the gel layer (up to 8 major aggregates/well) and allowed to attach by incubating at 37°C for 46 h. To assess the effect of inhibitors, cell aggregates were preincubated in SB203580, WP631, and SP600125 during the attachment period. The supernatant culture medium was removed, and 0.3 ml of fibrin solution containing the experimental reagent(s) was added on top of the aggregates to form the overlying fibrin gel. The culture was incubated at 37°C for 30 min to ensure polymerization. Serum-free medium supplemented with
-aminocaproic acid and aprotinin, as indicated above, plus the reagent(s) under study were added to each well (0.3 ml/well). For concentrations of reagents used, see RESULTS.
The culture medium was replaced with supplemented fresh medium every 24 h. In each experiment, multiple wells (26 wells) were used for each experimental condition. The data presented in this report were obtained using BAEC derived from a local slaughterhouse, and the cells were between passages 6 and 8. Similar results were obtained using two different batches of BAEC purchased from Clonetics (San Diego, CA), between passages 3 and 5. In addition, multiple batches of recombinant TGF-1 purchased from Sigma and R&D Systems exhibited similar effects. The culture medium from multiple wells was pooled for analysis of enzyme activity. Images of tubular structures were taken using a SPOT INSIGHT QE camera attached to a Nikon microscope at a magnification of x100.
Image analysis. Regions of the images of the tubular structures (400 x 400 pixels) were selected for analysis of directionality. A MatLAB script designed for this study was used to process the images. The script makes use of the Canny method for edge diction to separate cells from the background (7) and the MatLAB function bwlabed to identify objects in an image on the basis of connectivity of the pixels. The function bwlabed, when used in conjunction with the function regionprops, is able to calculate values for eccentricity and orientation of each object identified in an image. The values of eccentricity, which are a measure of linearity with a value of 0 being a circle and a value of 1 being a line segment, were averaged for all objects within the image. The orientation of the objects (i.e., the angle) relative to the x-axis was measured in degrees, and the standard deviation of all the angles determined for the objects in a given image was used as a measure of orientation of the structures. The logic behind this index is that if all objects are oriented in the same direction, the standard deviation will be low, and if the objects are not oriented in one direction, the standard deviation will be increased. In addition, the fractal dimension of each image was calculated using the box counting method (3, 30). Fractal dimension is a measure of the space-filling ability of a pattern. All P values were calculated using a two-tailed Students t-test.
Zymographic analysis of conditioned medium.
Casein zymography was performed on 10% or 12% SDS-polyacrylamide gels containing 2 mg/ml -casein (Sigma Chemical) and 34 µg/ml plasminogen (American Diagnostica, Stamford, CT). After electrophoresis, the gel was washed twice (30 min each) in 2.5% Triton X-100 and incubated in zymography buffer (50 mM Tris·HCl, pH 8.0, and 10 mM CaCl2) for 2448 h. Caseinolytic activity was visualized as a clear zone with Coomassie Brilliant Blue R-250 staining.
Gelatin zymography was performed on 10% SDS-polyacrylamide gels containing 0.1% gelatin (Sigma Chemical). To perform reverse gelatin zymography, 30 ng/ml pro-matrix metalloproteinase (MMP)9 (Calbiochem) were added to the gel. After a washing in 2.5% Triton X-100, the gels were incubated overnight in zymography buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2, and 0.2% NaN3). Gelatinolytic and inhibitory activity was visualized as a clear or dark zone, respectively, with Coomassie Brilliant Blue R-250 staining.
Northern blotting. Total RNA from BAEC was prepared using TriReagent solution (Sigma Chemical). For Northern blot analysis, RNA samples (10 µg/lane) were fractionated by electrophoresis on a 1.3% agarose formaldehyde gel and transferred to GeneScreen Plus membrane (NEN Life Science Products, Boston, MA). Hybridization with 32P-labeled probes was performed at 68°C for 1.53 h in Quickhyb solution (Stratagene, La Jolla, CA). After hybridization, filters were washed twice at room temperature in 2x SSC, 0.1% SDS for 510 min each at 5568°C, and in 0.1x SSC, 0.1% SDS for 1530 min at room temperature, and subjected to autoradiography. All cDNA probes were prepared by random primer labeling followed by purification using a Sephadex G-50 spin column (Roche Molecular Products, Alameda, CA).
The probe used to detect TSP1 was a 675-bp XmnI enzyme fragment of murine TSP1 cDNA. To detect PAI-1 and urokinase-type (u) plasminogen activator (PA) mRNAs, cDNA probes were generated by RT-PCR with the use of total RNA isolated from BAEC as a template. The primers used for RT-PCR amplification were as follows: 1) for PAI-1, the 5'-primer TCATTCCCAAATTCTCCAGC and the 3'-primer CAACGTGGTTTTCTCACCCT; 2) for uPA, the 5'-primer ACAATCCCAGTCAGGGTCAG and the 3'-primer AGGTCACCAACACCGAGAAC. RNA loading was verified using cDNA probes specific for the glyceraldehyde phosphate dehydrogenase (GAPDH) and the acidic ribosomal phosphoprotein PO (36B4) mRNAs.
Western blotting. Protein extracts were fractionated on 10% SDS-PAGE. After transfer to polyvinylidene difluoride (PVDF) membrane (PerkinElmer Life Sciences, Wellesley, MA) overnight at 24 V, the membrane was rinsed twice (5 min each) in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) and blocked for 1 h with 0.2% nonfat milk in TBS containing protease and phosphatase inhibitor cocktails. The membrane was then incubated with rabbit anti-full-length JNK (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phospho-JNK (Promega, Fitchburg, WI) in 0.2% nonfat milk in TBS containing 0.1% Tween 20 and protease and phosphatase inhibitor cocktails. The membrane was washed and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). The signal was detected by use of a typhoon scanner.
In vitro kinase assays.
BAEC cultured in 100-mm-diameter dishes were treated with serum-free medium containing the JNK inhibitor SP600125 (10 µM) up to 40 min. JNK activity assays were performed as described previously (24). At the indicated times, the culture plates were placed on ice. Cell monolayers were washed with cold PBS, and extracts were prepared in lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM -glycerophosphate, 1 mM Na3VO4, and EDTA-free protease inhibitor cocktail from Roche). The cell lysate was centrifuged for 15 min at 14,000 g, and the supernatant was retained for kinase assays. Because JNK has been identified as the only known kinase capable of phosphorylating c-Jun, total cell extracts were used for JNK activity assays. Aliquots containing 150 µg of total cellular protein were incubated with an equal volume of kinase buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 5 µM ATP, and 10 µCi [
-32P]ATP) and 2 µg of glutathione S-transferase (GST)-c-Jun(189) (Cell Signaling, Beverly, MA) at 37°C for 30 min. Glutathione-Sepharose 4B (40 µl) was added to the reaction mix, and samples were rotated for 30 min at 4°C. Pellets were washed three times with PBS containing 1% Triton X-100 and resuspended in Laemmli sample buffer. The samples were heated at 95° for 4 min and fractionated on 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, Billerica, MA). To detect phosphorylated c-Jun(189), the blot was exposed to Kodak X-AR film. Total GST-c-Jun protein on the membrane was visualized by staining with Ponceau S dye (Sigma).
Assessment of cell cytotoxicity and migration. The effect of SB203580, WP631, and SP600125 treatment on BAEC cytotoxicity and migration was examined using a cytotoxicity detection kit (Promega) and an in vitro wounding assay, respectively. To assess cell cytotoxicity, BAEC harvested from a confluent culture were seeded in a 96-well plate precoated with fibrinogen (20,000 cells/well). The cells were incubated for 24 h to allow attachment. The medium was replaced with serum-free medium containing various concentrations of the inhibitors. After 24-h incubation, cell cytotoxicity assays were performed according to the manufacturers protocols. Fluorescent detection of resorufin produced by a coupled enzymatic reaction involving lactate dehydrogenase released by lysed cells was used to assess cytotoxicity.
Cell migration was investigated using the in vitro wounding assay. BAEC were seeded in a six-well dish (4 x 105 cells/well) and incubated overnight to allow attachment. Before scraping, the cell monolayers were preincubated for 1 h in serum-free medium containing the inhibitors and manually scraped with a pipette tip. The cultures were further incubated for 24 h. Digital images of the wounded areas were captured using an inverted microscope (x4 objective) immediately after wounding and 24 h later. The area of the wound within a defined image frame (450 x 800 pixels) was determined using the Spot software and used to calculate percent wound area closure as follows:
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RESULTS |
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MatLAB programming was used to process the images of the tubular structures (Fig. 1B). Analysis of the images (Fig. 1C) showed that the eccentricity (linearity) value (mean ± SE) determined for TGF-1-treated cells was higher than for control cells [TGF-
1: 0.9211 ± 0.0071, no. of images (n) = 5; control: 0.87102 ± 0.006417, n = 5; P = 0.00079]. On the other hand, the standard deviation of orientation (angle variation) (Fig. 1D) of TGF-
1-treated cells was significantly lower than for control cells (TGF-
1: 25.61 ± 1.04, n = 4; control: 48.95 ± 1.99, n = 5; P = 0.00003), showing that TGF-
1 treatment induced formation of more linear structures oriented in a similar direction. Analysis of fractal dimension (space filling) (Fig. 1E) also showed that the TGF-
1-treated cells had a more space-filling pattern than the control cells (TGF-
1: 1.638 ± 0.0118, n = 5; control: 1.590 ± 0.0074, n = 4; P = 0.0153). These results are in agreement with the conclusion that TGF-
1 treatment induced a bipolarized capillary tubelike pattern.
The TGF-1-induced pattern was more obvious in areas of high density of outgrowths and where outgrowths from neighboring cell aggregates anastomosed. These observations and the known stimulatory effect of TGF-
1 on extracellular matrix protein production suggest that localized secretion of proteins from opposite ends of the tubular structures generates a chemoattractant concentration gradient for other cells. Alternatively, it is also possible that the altered pattern of tubular structures suggests that a change in the cellular microenvironment due to a localized increase in matrix protein expression contributes to the vessel directionality observed.
Inhibition of uPA activity in the presence of TGF-1.
TGF-
1-induced alteration of uPA/PAI-1 mRNA expression has been implicated in inhibition of lumen formation in vitro (43). To study whether regulation of the PA and MMP systems was associated with the change in capillary tube patterning, conditioned medium was analyzed for select enzymes and their respective inhibitors.
To determine whether the TGF-1-induced change in capillary tube patterning was dependent on membrane type (MT)1-MMP-mediated pericellular fibrinolysis, conditioned medium collected from cells cultured in the absence or presence of TGF-
1 was analyzed for the presence of MMP2 by gelatin zymography. An increase in the amount of active MMP2 would have been indicative of an increase in MT1-MMP activity (32). As shown in Fig. 2, the latent form of MMP2 (proMMP2) was expressed at a high level. However, the level of active MMP2 (bottom band) detected in TGF-
1-treated samples was indistinguishable from that detected in control and FGF2-treated samples. It is also unlikely that the higher level of MMP9 detected in the presence of TGF-
1 (Fig. 2A) mediated the morphogenetic change. Although the level of MMP9 was higher in FGF2-treated samples than in control samples, the capillary tube pattern remained random. Similarly, no significant alterations in tissue inhibitor of metalloproteinases (TIMP)2 and TIMP3 expression levels were detected when conditioned medium from control and TGF-
1-treated BAEC was analyzed either by reverse gelatin zymography (Fig. 2B) or by Western and Northern blotting analysis (unpublished observation). These results suggested that the MMPs analyzed did not play a required role in TGF-
1-induced capillary tube patterning.
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DISCUSSION |
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Fibrin matrix, like other macromolecular matrices, has been used to study in vitro the morphogenesis of endothelial cells into capillary-like tubes. Endothelial cell monolayers sandwiched between two fibrin gels (8), endothelial cells incorporated in or seeded on top of a fibrin matrix (15, 56), and cell-coated microcarrier beads incorporated in a fibrin matrix (37) have been shown to give rise to capillary-like structures. On the basis of studies using fibronectin, collagen, matrigel, and fibrin matrices, it has been proposed that traction forces exerted by cells on viscoelastic substrata induce reorganization of the vicinal matrix into cords that provide positional information for the development of capillary-like structures (13, 26, 37, 57, 58). In the present study, capillary tube patterning by endothelial cell aggregates sandwiched between two layers of fibrin matrix was studied. Although this method is complex and difficult to visualize and quantify, because capillary-like tubes are formed in multiple directions and at different surface layers, it offers unique advantages: 1) Endothelial cell aggregates have been shown to remodel over time to establish a differentiated surface layer of endothelial cells and a center of unorganized endothelial cells that, if not rescued by survival factors, subsequently undergoes apoptosis. The cells on the surface become quiescent and establish firm cell-to-cell contacts and can be induced to express differentiation antigens such as CD34 in response to vascular endothelial growth factor (VEGF) (31). 2) Essentially all the endothelial cells growing into the surrounding fibrin matrix participate in capillary-like tube formation. Analysis of the changes in the levels of proteins secreted into the supernatant medium (e.g., PA activity) reflects changes in gene regulation during the angiogenic process.
The capillary tubes developed radially along the interface between the two layers of fibrin matrix. The presence of TGF-1 induced a more planar pattern of endothelial cell morphogenesis compared with control or FGF2-treated cells. Because TGF-
1 has been shown to induce FGF2 expression in mouse embryo fibroblasts (46), other investigators suggested that the guided migration of endothelial cells along a preestablished capillary-like structure is regulated by FGF2 (38). It is possible that in the current study FGF2 did contribute to the TGF-
1-induced capillary tube pattern. However, if FGF2 was the major factor mediating the phenotypic change, exogenous FGF2 should have reversed the TGF-
1-induced capillary tube pattern. When FGF2 and TGF-
1 were added simultaneously, the resulting capillary-like pattern was similar to the structured pattern observed in the presence of TGF-
1 alone and not the random pattern observed in the presence of FGF2 alone. These observations suggested that the observed TGF-
1-induced capillary tube pattern was not mediated by FGF2.
TGF-1 is a widely expressed pleiotropic cytokine controlling cellular functions that are critical to animal embryo development and tissue homeostasis. Despite the diversity and physiological importance of the TGF-
-elicited cellular responses, the basic signaling cascade consists of two receptor serine-threonine protein kinases (receptor types I and II) and a family of receptor substrates (the SMAD proteins) that translocate into the nucleus and induce or suppress target gene expression (33). In this report, TGF-
1 activated the basic TGF-
/SMAD signaling pathway in BAEC sandwiched in fibrin matrix as evidenced by 1) the inhibition of uPA activity by upregulation of PAI-1 expression and 2) the reversal of the inhibition of uPA activity by the p38 MAPK inhibitor SB203580. Moreover, the DNA intercalator WP631 and the cytokine TNF-
have been shown to inhibit SMAD 3/4-induced collagen synthesis (19). Because of the ability of WP631 and TNF-
to antagonize TGF-
1-induced capillary tube pattern, it is likely that SMAD signaling is involved in capillary tube patterning. Furthermore, in support of the role of the SMADs in morphogenesis, transfection with Smad 2 and Smad 4 has been shown to induce fibroblast-myofibroblast terminal differentiation (17).
Although the majority of in vitro studies have found TGF-1 to inhibit endothelial cell functions, including tube formation, it has also been shown to regulate capillary tube formation in a biphasic manner. In an in vitro model of angiogenesis in which bovine microvascular endothelial cells from adrenal cortex were seeded on the surface of a matrix, VEGF- or basic FGF-induced invasion of collagen or fibrin gels was further induced when TGF-
1 was co-added to the system at 0.23 ng/ml and inhibited when TGF-
1 was added at 10 ng/ml. In addition to the effect of TGF-
1 on invasion, lumen size in the resulting structures was progressively reduced with increasing concentrations of TGF-
1 (42). In the present study, the lowest concentration of TGF-
1 that reproducibly altered the random pattern of capillary tubes formed was 5 ng/ml. Further studies are needed to determine the specific role of the different TGF-
receptors in our system. Although conflicting, more recent studies (21, 40) suggested that the TGF-
type I receptors ALK1 and ALK5 transduce TGF-
-dependent signals that regulate the activation and resolution phases of the vessel formation process and that differential regulation of TGF-
type I receptors might explain the biphasic effect of TGF-
1.
Alternatively, a change in the net balance of extracellular matrix proteolysis has been proposed as the mechanism of TGF-1-dependent inhibition of endothelial cell invasion of amniotic basement membrane and three-dimensional collagen or fibrin gels (35, 36, 41, 43, 44, 51). MMPs, in particular MT1-MMP, have been shown to be critical for invasion of fibrin gels and formation of capillary-like structures by tissue explants and micro- and macrovascular endothelial cells (11, 23, 32). The results reported in this study do not indicate that the MMPs analyzed play a direct role in capillary tube patterning. To investigate the possible role of uPA enzyme activity and TSP1 expression in capillary tube patterning, the p38 MAPK inhibitor SB203580 was used. SB203580 inhibited TGF-
1-induced upregulation of PAI-1 and TSP1 expression but not TGF-
1-induced capillary tube patterning. Thus differential expression of TSP1, uPA, MMP2, MMP9, and possibly MT1-MMP as inferred from the pattern of active MMP2 enzyme levels or their respective inhibitory regulators, PAI-1 and TIMP, could not account for the TGF-
1-induced capillary tube pattern. These results suggested that TGF-
1-elicited signals regulate capillary tube patterning through downstream signaling events that are distinct from the cascades that control the PA system.
Another interesting and important observation is the role of the JNK pathway in cell survival and capillary tube patterning. Although more studies remain to be done, this study shows that intracellular signals, and in particular the JNK pathway, play a key role in BAEC morphogenesis in fibrin matrix. Inhibition of JNK activity by SP600125 prevented capillary tube formation and wound closure. In the presence of TGF-1, SP600125 was less inhibitory because the JNK pathway functions within the overall context of the state of activation of other signaling pathways (14). Although it is established that JNK contributes to some apoptotic responses, JNK may also contribute to survival signaling (1) under the defined serum-free culture conditions in this study. Additionally, in support of our results using endothelial cells, the JNK pathway has been shown to regulate fibroblast motility (28). The above-mentioned functions of the JNK pathway are consistent with genetic and overexpression studies in Drosophila and Xenopus that have shown that JNK signaling coordinates dorsal closure (34), planar cell polarity (6), and convergent extension (61). The JNK pathway is thought to affect both the cytoskeleton and gene expression. Interestingly, the TGF-
family member decapentaplegic is one target gene of the JNK pathway that functions as a chemoattractant to more lateral cells and thereby induces coordinated cell elongation and movement during dorsal closure (34). In conclusion, the observations of this report and cited studies indicate that the TGF-
and JNK signaling pathways play important roles in capillary tube patterning and morphogenesis. TGF-
1 modulates PAI-1 and TSP1 expression, both of which are important regulators of angiogenesis. TGF-B1-induced alteration of capillary tube patterning is mediated through a signaling pathway that is independent of its effect on PAI-1 and TSP1 expression.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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