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Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea
1Correspondence: Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Daejeon, 305-701, Republic of Korea. E-mail: gykoh{at}kaist.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Ang3 • Ang4 • Ang1 • Tie2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6)
. Ang1 and Ang2 share 
60% amino acid identity and bind with similar affinity to the endothelial cell tyrosine kinase receptor Tie2 (1
, 2)
. Ang1 and Ang2 have characteristic protein structures that contain coiled-coil domains in the amino-terminal region and a fibrinogen-like domain in the carboxyl-terminal region (1
, 2)
. In vivo analyses by targeted gene inactivation of Ang1 and transgenic overexpression of Ang2 have revealed that Ang1 recruits and sustains peri-endothelial support cells (7)
whereas Ang2 disrupts blood vessel formation in the developing embryo by antagonizing the effects of Ang1 on Tie2 (2)
. Thus, Ang2 was originally thought to be a naturally occurring antagonist of Ang1 that competes for binding to Tie2 and blocks Ang1-induced Tie2 autophosphorylation (2)
. However, recent data suggest that Ang2 seems to have context-specific effects, activating Tie2 on some cells while blocking Tie2 activation on other cells or under different conditions (2
, 8
9
10)
. A study of Ang2 knockout mice revealed that Ang2 is dispensable for embryonic vascular development but is required for subsequent angiogenic remodeling (10)
. Unexpectedly, mice lacking Ang2 exhibit major lymphatic vessel defects (10)
.
Through low stringency hybridization screening with Ang1 and Ang2 cDNAs, mouse angiopoietin-3 (Ang3) and human angiopoietin-4 (Ang4) were identified (11)
. Ang3 and Ang4 are interspecies orthologs and represent a third protein that binds to the Tie2 receptor (11)
. Ang4 phosphorylates Tie2 whereas Ang3 inhibits Ang1-induced phosphorylation of Tie2 in human endothelial cells (11)
. Thus, Ang4 has been thought to be an agonist of Tie2 whereas Ang3 has been thought to be an antagonist of Tie2. Although the expression pattern of Ang4 varies among various human tumors (12
13
14)
, hypoxia or endothelial cell growth factors lead to increasing levels of Ang4 expression in a human glioblastoma cell line and endothelial cells (15
16
17)
. Ang3 expression is increased in the lung, liver, cerebellum, and heart in response to hypoxia in rats (18)
. Thus, regulation of Ang3 and Ang4 expression could be involved in physiological and pathophysiological angiogenesis. However, the biological effects of Ang3 and Ang4 have not been studied. Therefore, we examined the effect of Ang3 and Ang4 in primary cultured human umbilical vein endothelial cells (HUVECs), in mouse lung microvascular endothelial cells (MLMECs), and in vivo using the mouse corneal micropocket assay. We found that Ang3 acts as an agonist to Tie2 on mouse endothelial cells whereas Ang4 acts as an agonist to Tie2 on human endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and mouse hepatic fibrinogen/angiopoietin-related protein (HFARP) (20)
. All recombinant plasmids were constructed using standard molecular cloning methods.
Transfection and purification of the recombinant proteins
Recombinant proteins were obtained by transient expression in COS-7 cells (American Type Culture Collection, Manassas, VA, USA) using Effectene liposomal transfection according to the manufacturer’s instructions (Qiagen, Inc., Hilden, Germany). Transfection efficiency of the genes was 30–40%. The supernatant was harvested from transfected cells after 48–96 h. Recombinant proteins containing the FLAG sequence were purified by column chromatography on anti-FLAG M1 antibody agarose affinity gel (Sigma-Aldrich, Inc., St. Louis, MO, USA). After purification of COS-7 supernatants, recombinant proteins were quantitated using the Bradford assay and confirmed with Coomassie blue staining of an SDS-PAGE gel. The yield of each recombinant protein was 800–1000 µg per liter of COS-7 cell supernatant.
Characterization of the recombinant proteins
SDS-PAGE analyses of proteins were performed under nonreducing and reducing (10 min at 95°C in 0.435 M ß-mercaptoethanol) conditions. Binding of the recombinant proteins to the soluble extracellular domain of Tie1-Fc (sTie1-Fc, T1) or Tie2-Fc (sTie2-Fc, T2) (R&D Systems, Minneapolis, MN, USA) was assayed using an in vitro binding assay. Each recombinant protein (20 ng) was mixed with 100 ng of sTie1-Fc or sTie2-Fc and incubated in 500 µL Tris buffer solution (50 mM Tris, 100 mM NaCl, pH 7.4) containing 0.02% TritonX-100 at 4°C for 2 h. Then, 20 µL of protein-A agarose beads (Oncogene, San Diego, CA, USA) was added and incubated for another 1 h at 4°C. The protein-A conjugated samples were washed twice with 1 mL of Tris buffer containing 0.02% TritonX-100. Samples were eluted with sample buffer and heat-denatured. The samples were further separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, and probed with mouse anti-FLAG M1 antibody.
Cell culture
HUVECs were prepared from human umbilical cords by collagenase digestion and maintained as described previously (21)
. The primary cultured HUVECs used in this study were between passages 2 and 3. MLMECs were prepared according to Tiruppathi et al. (22)
with slight modification. Immediately after the mice (2 wk old) were killed, lung tissues were removed, washed with Ca2+- and Mg2+-free PBS containing 1% antibiotics and antimycotics, and minced into small pieces. The minced tissue was suspended in 30 mL of Hank’s buffered salt saline (Sigma-Aldrich) containing 1% collagenase and 0.5% bovine serum albumin (BSA), and digested with stirring for 60 min at 37°C. The digest was filtered through 100 µm nylon mesh (BD Bioscience, Bedford, MA, USA) and centrifuged at 2000 rpm for 5 min. Cells were washed with M-199 medium containing 10% fetal bovine serum (FBS), resuspended in suspension buffer with 1.5 µg/mL rat anti-mouse PECAM-1 antibody and Dynabeads M-450 sheep anti-rat IgG antibody (Dynal Biotech, Inc., Lake Success, NY, USA), and incubated for 60 min at 4°C on a rotator. After incubation, MLMECs were selected using a magnetic particle concentrator (Dynal MPC®-1 column, Dynal Biotech) and plated onto 60 mm cell culture dishes coated with 2 µg of human fibronectin (BD Biosciences). These MLMECs were maintained in M-199 medium containing 20% heat-inactivated FBS at 37°C in 5% CO2. The endothelial origin of the cultures was confirmed by the presence of PECAM-1 detected with rat anti-mouse PECAM-1 antibody (BD Biosciences) by immunofluorescence. The primary cultured MLMECs used in this study were between passage 4 and passage 5.
Biochemical assays
The confluent cells were starved in M-199 medium with 1% FBS for 16 h before addition of Ang3, Ang4, or Ang1. Tie2 and Akt (S473) phosphorylation assays were performed as described (21
, 23)
using HUVECs, MLMECs, and lung tissue from mice (FVB males, 12 wk old). Briefly, to measure the phosphorylation of Tie2, 0.5 mg of HUVECs lysate protein or 1.0 mg of lung lysate protein was immunoprecipitated with goat anti-human Tie2 antibody (R&D Systems). Immunoprecipitates were Western blotted with anti-phospho-tyrosine antibody, and the membrane was reblotted with rabbit anti-human Tie2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) to verify equal loading of protein in each lane. To measure Akt (Ser473) phosphorylation, the treated HUVECs or mouse lung tissues were washed twice with PBS, dissolved in sample buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% SDS, 1% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 µg/mL aprotinin, 1 µg/mL pepstatin, and 1 µg/mL leupeptin). Protein lysates (50 µg) were boiled, separated by SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were treated and the phosphorylation levels analyzed according to the manufacturer’s protocol (Cell Signaling, Beverly, MA, USA). After immunoblot analysis with rabbit anti-phospho-Akt (Ser473) antibody, the membrane was stripped and reincubated with rabbit anti-Akt antibody to detect total Akt protein. All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film, Tokyo).
Biological assay
The apoptosis survival assay was performed as described (21
, 24)
. Briefly, to examine survival from apoptosis, HUVECs were plated onto gelatinized 24-well plates (7x104 cells/well) in M-199 containing 20% FBS and incubated for 12 h. Then the wells were extensively washed with PBS and the medium was changed to serum-free M-199 containing the indicated concentration of Ang3, Ang4, or Ang1 for 30 h. Floating cells were collected with two washes in PBS. Adherent cells were collected by trypsinization. All cells were stained with the annexin-V-fluos staining kit (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at 20°C. After staining of annexin-V and propidium iodide, cells were analyzed on a flow cytometer and data were analyzed with CellQuest software (Becton Dickinson). Adherent apoptotic cells from part of each sample were detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (Oncor, Gaithersburg, MD, USA) to confirm results obtained from flow cytometry. The migration assay in HUVECs was performed using a modified Boyden chamber (Neuroprobe, Inc., Cabin John, MD, USA) as described previously (24
, 25)
. Briefly, the indicated concentration of Ang3, Ang4 or Ang1 in M199 containing 1% BSA were placed in the bottom wells of the chamber. Polycarbonate filters with 8 µm pores (Poretics Corp., Livermore, CA, USA) were coated with 50 µg/mL fibronectin/0.2% gelatin and placed between the test substances and the upper chambers. Cells were trypsinized, washed twice in M199, and resuspended in M199 containing 1% BSA. We placed 2 x 105 cells into each well in the upper chamber, then incubated for 6 h at 37°C in a humidified chamber with 5% CO2. After incubation, the nonmigrated cells were removed from the upper side of the filters with a cotton ball. The filters were fixed with methanol, mounted onto microscope slides, and stained with Diff-Quik solution. The migrated cells were counted at 100x magnification using a microscope.
Animals
Male FVB mice (Jackson Labs, Bar Harbor, Maine), 10–12 wk old, were used for all experiments, except as noted. All animal care and experimental procedures were performed with approval from the Animal Care Committees of KAIST.
Mouse corneal angiogenesis assay
Eight-wk-old male C57BL/6J mice (Jackson Labs, Bar Harbor, ME, USA) were used. After systemic and local eye anesthesia, a central, intrastromal linear keratotomy of 0.6 mm was performed with a surgical blade, and a micropocket was dissected toward the temporal limbus using a modified von Graefe knife (26)
. A sucrose aluminum sulfate pellet was coated with hydron polymer containing one of the following: control buffer, Ang3 (600 ng/pellet), Ang4 (600 ng/pellet), ARP1 (600 ng/pellet), or HFARP (600 ng/pellet). The pellet was positioned 0.6–0.8 mm from the corneal limbus. On postoperative day 8, the arc of corneal circumference occupied by angiogenesis (circumferential angiogenesis, in degrees) and vessel lengths and numbers were measured using a surgical stereomicroscope (SV6, Carl Zeiss, Gottingen, Germany) equipped with an AxioCAM CCD camera (Carl Zeiss, Germany).
Data analyses
Data are expressed as mean ± standard deviation (SD). Statistical significance was tested using 1-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P < 0.05 or P < 0.01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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68 kDa, 72 kDa, 70 kDa, and 68 kDa, respectively (Fig. 1
A). Under nonreducing conditions, Ang3, Ang4, and Ang2 mostly formed characteristic disulfide-linked dimers whereas Ang1 mostly formed characteristic disulfide-linked multimers of various sizes. Ang3, Ang4, and Ang2 rarely formed variable higher order multimers.
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Ang3 and Ang4 bind to Tie2
In vitro binding assays revealed that Ang3 and Ang4 bound to sTie2-Fc but not to soluble sTie1-Fc (Fig. 1B
). Approximately 92%, 85%, 74%, and 95% of the total protein amount of Ang3, Ang4, Ang1 and Ang2, respectively, bound to sTie2-Fc (Fig. 1B
).
Ang4, but not Ang3, strongly induces Tie2 and Akt phosphorylations in HUVECs
In HUVECs, Ang3 (400 ng/mL) slightly increased Tie2 phosphorylation for 2 h and Ang4 (400 ng/mL) markedly increased Tie2 phosphorylation for 2 h (Fig. 2
A, B). Ang4 increased Tie2 phosphorylation as early as 10 min and produced a maximal effect at 30 min. These effects declined, but remained above control levels, at 4 h. The maximum mean increases in Tie2 phosphorylation by Ang3 and Ang4 were 1.7-fold and 7.8-fold. Tie2 phosphorylation evokes several signaling pathways (4
5
6)
. Of these, Akt phosphorylation (Ser473) is the main downstream signaling of Tie2 (4
5
6)
. We examined the effect of Ang3 and Ang4 on Akt phosphorylation (Ser473) in HUVECs. Whereas Ang3 (400 ng/mL) produced almost no effect on Akt phosphorylation for 4 h, Ang4 (400 ng/mL) strongly increased Akt phosphorylation for 4 h (Fig. 2C, D
). The pattern of Ang4-induced Akt phosphorylation was similar to the pattern of Ang4-induced Tie2 phosphorylation (Fig. 2C, D
). The maximum mean increase in Akt phosphorylation by Ang4 was 3.6-fold. Thus, Ang4, but not Ang3, is a strong agonist of the Tie2-Akt system in HUVECs.
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Ang4, but not Ang3, strongly induces survival and migration in HUVECs
The Ang1-Tie2-Akt system is mainly involved in endothelial cell survival and migration (4
5
6
, 21
, 27
, 28)
. Therefore, we examined the effect of Ang3 and Ang4 on survival and migration in HUVECs. Consistent with the biochemical effects observed, addition of Ang4 (400 ng/mL) produced a strong survival effect during serum deprivation in HUVECs, evidenced by fewer floating and more adherent cells seen with phase-contrast microscopy and fewer positively stained nuclei seen in cells assayed with TUNEL vs. cells treated with control buffer at 24 h (Fig. 3
A). Flow cytometry analyses also indicated that Ang4 (400 ng/mL) produced 54% greater survival 30 h after serum deprivation (Fig. 3B
). In comparison, addition of Ang3 (400 ng/mL) produced a slight survival effect. After 6 h treatment of HUVECs, Ang4 (400 ng/mL) increased migration 2.1-fold whereas Ang3 (400 ng/mL) produced almost no effect on migration (Fig. 3C
). Thus, the Ang4-induced survival and migration effects in human endothelial cells agree with those recently reported (17)
. These effects were similar to the survival and migration effects of Ang1 (400 ng/mL) (Fig. 3B, C
). Thus, Ang4, but not Ang3, is a strong survival and migratory factor in HUVECs.
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Ang3, but not Ang4, strongly induces Tie2 and Akt phosphorylations in mouse lung
To examine the effect of Ang3 and Ang4 on phosphorylation of Tie2 and Akt in vivo, we used samples from the lung because examination of tissue expression of Tie2 in adult mouse revealed that the protein is most abundant in microvascular lung endothelial cells (data not shown). Intravenous injection of Ang3 (30 µg) increased Tie2 and Akt phosphorylation at 15 min 8.3-fold and 3.1-fold, respectively; intravenous injection of Ang4 (30 µg) increased these phosphorylations 3.1-fold and 2.2-fold, respectively. These phosphorylation levels then gradually decreased at 30 min and 60 min. Thus, unexpectedly, Ang3 induced phosphorylation of Tie2 and Akt, with both greater magnitude and persistence than that of Ang4 (Fig. 4
). Thus, Ang3 is more potent than Ang4 for activation of Tie2 in vivo in mouse lung microvascular endothelial cells.
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Ang3 is more potent than Ang4 in Akt phosphorylation and survival in primary cultured MLMECs
We thought that the differential effects of Ang3 and Ang4 on Tie2 and Akt in HUVECs could have been the result of the species difference, so we examined the effect of Ang3 and Ang4 on Akt phosphorylation and survival in primary cultured MLMECs (Fig. 5
A). Indeed, Ang3 was more potent than Ang4 in Akt phosphorylation and survival in these cells (Fig. 5B-D
). The Ang3-induced survival effect was similar to the Ang1-induced survival effect. Thus, Ang3 acted as a potent agonist to Tie2 in mouse lung endothelial cells.
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Both Ang3 and Ang4 induce strong angiogenesis in vivo
To assess the effect of Ang3 and Ang4 on in vivo angiogenesis, we performed a mouse corneal micropocket assay with implantation of pellets containing the recombinant proteins. On the eighth day after pellet implantation, corneal angiogenesis was evaluated using a stereomicroscope. The pellets containing either Ang3 or Ang4 induced strong angiogenesis but control buffer did not induce significant angiogenesis (Fig. 6
A). To exclude a nonspecific action of protein subjected to the purification procedure, we performed corneal angiogenesis with the pellet containing either ARP1 or HFARP, which are similar to angiopoietins but do not have angiogenic activities. The pellets containing Ang3 or Ang4 induced corneal angiogenesis extending from the limbus across the cornea with an increased number of vessels and increased degrees of arch (Fig. 6B-D
). Although mouse Ang3 appeared to be slightly more effective than human Ang4 in this assay, there was no statistically significant difference between them. Neither ARP1 (19)
nor HFARP (20)
induced significant angiogenesis by the 8th day after pellet implantation. Thus, both Ang3 and Ang4 showed a potent and specific angiogenic effect in vivo in mouse.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Using a mammalian cell expression system, we generated recombinant protein for both proteins. Ang3 and Ang4 formed disulfide-linked dimers. Our findings with in vitro and in vivo experiments indicated that Ang3 does not act as an agonist to human Tie2 whereas Ang4 acts as a strong agonist to human Tie2. In comparison, Ang3 acts as a strong agonist to mouse Tie2 whereas Ang4 acts as a moderate agonist to mouse Tie2. Our findings with Ang3 are different from the original description of Ang3 (11)
, which showed Ang3 to be an antagonist to Tie2. Thus, like Ang4, Ang3 is an effective agonist rather than antagonist for Tie2 but is effective only in its native species (Fig. 7
).
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Although Ang3 (mouse) and Ang4 (human) are interspecies orthologs, the percent amino acid identity between them is only 65% (11)
; the percent amino acid identity between human Ang1 and mouse Ang1 is 99% and between human Ang2 and mouse Ang2, 87% (1
, 2)
. Although the percent amino acid identity between Ang3 and Ang4 is much less than other human and mouse counterparts, their chromosomal localizations indicate that the Ang3 locus in mouse is indeed syntenic to the Ang4 locus in humans (11)
. Amino acid sequence analyses of Ang3 and Ang4 reveal that, like Ang1 and Ang2, Ang3 and Ang4 have characteristic protein structures that contain coiled-coil domains in the amino-terminal region and a fibrinogen-like domain in the carboxyl-terminal region (1
, 2
, 11)
. As described previously (29
, 30)
, our data indicate that the majority of Ang1 forms disulfide-linked variable multimers whereas the major form of Ang2 is a disulfide-linked dimer. Like Ang2, the major form of Ang3 and Ang4 is the disulfide-linked dimer. These similarities and differences among the angiopoietin proteins may result from the number and location of conserved cysteine residues (1
, 2
, 11)
. The cysteines in Ang3 and Ang4 more closely resemble those in Ang2 than those in Ang1 (1
, 2
, 11)
. Our in vitro binding experiments indicated that Ang3 and Ang4 have similar binding characteristics to human Tie2. Similar to an earlier report (11)
, our biochemical analyses indicated that Ang3 did not produce any notable effect on human Tie2 and Akt phosphorylations whereas Ang4 strongly phosphorylated human Tie2 and Akt. In comparison, Ang4 moderately phosphorylated mouse Tie2 and Akt whereas Ang3 strongly phosphorylated mouse Tie2 and Akt.
The results regarding strong Ang3-induced phosphorylations of mouse Tie2 and Akt conflict with those presented by Valenzuela et al. (11)
, who reported that Ang3 had almost no effect on Tie2 phosphorylation in human and mouse endothelial cells. The difference between our results and Valenzuela’s results (11)
may have resulted from the use of different Ang3 recombinant proteins and different cell types. They used chimeric Ang3 fusion protein (dimeric Ang3FD-Fc), which is a fusion of the fibrinogen-like domain of Ang3 to the constant region (Fc), whereas we used native Ang3 recombinant protein. A recent report (30)
indicated that angiopoietin-induced Tie2 clustering and oligomerization could be important for angiopoietin-induced Tie2 phosphorylation. Ang3FD-FC-induced Tie2 clustering and oligomerization could not properly occur whereas native Ang3-induced Tie2 clustering and oligomerization could properly occur. Thus, more than binding might be involved in Ang3-induced Tie2 and Akt phosphorylations. Therefore, a more detailed analysis of the molecular interactions among angiopoietins and Tie2 at the ligand receptor level should be made in order to clarify the real function of angiopoietins in vitro and in vivo. Valenzuela et al. used an endothelial cell line whereas we used primary cultured endothelial cells and Tie2-abundant lung tissues in vivo. Although our primary cultured MLMECs and lung tissues may not be a perfect representation of microvascular endothelial cells in vivo, they could be a more reliable representation of endothelial cells than are endothelial cell lines. Given these considerations, Ang3 is a potent agonist to mouse Tie2.
The Tie2-Akt system is involved mainly in endothelial cell survival, endothelial cell migration, and angiogenesis (4
5
6
, 31
32
33)
. Accordingly, Ang4 strongly induced the survival and migration of primary cultured human endothelial cells whereas Ang3 strongly induced the survival of primary cultured mouse endothelial cells. Moreover, both Ang3 and Ang4 produced strong angiogenesis in vivo. Basically, Ang4 is a potent agonist to human Tie2 whereas Ang3 is a potent agonist to mouse Tie2. Although recombinant Ang1 protein has potential therapeutic application in angiogenesis, endothelial cell survival, and prevention of vascular leakage (6
, 31
32
33
34
35
36
37)
, large-scale production of recombinant Ang1 is hindered by aggregation and insolubility of the protein. The activity of the protein frequently varies after purification. These difficulties are due to its multimeric structural characteristics. However, recombinant Ang4 forms a soluble dimer. Given that Ang4 has potency similar to Ang1, Ang4 may be an ideal alternative to Ang1 for therapeutic angiogenesis, therapeutic endothelial cell survival, and the prevention of vascular leakage in human diseases.
Whereas Ang1 mRNA is mainly present in mural cells (pericytes, smooth muscle cells) (1
, 7)
, Ang2 mRNA is present mainly in endothelial cells (2)
. However, the in situ tissue expression of Ang3 and Ang4 is unknown. Moreover, regulation of Ang3 and Ang4 expression is poorly known, although the regulation of Ang1 and Ang2 expression has been studied (6)
. Like Ang2, Ang3 and Ang4 expression levels increase in response to hypoxia, and endothelial cell growth factors lead to increasing levels of Ang4 expression in a glioblastoma cell line and endothelial cells (15
16
17
18)
. However, the molecular mechanisms of how Ang3 and Ang4 are regulated and the involvement of regulation of Ang3 and Ang4 expression in physiological and pathophysiological angiogenesis are unknown. Therefore, studies of in situ tissue expression of Ang3 and Ang4 and the regulation of Ang3 and Ang4 expression will be performed. In vivo analyses by targeted gene inactivation and transgenic overexpression of Ang3 would be warranted in order to clarify the role of Ang3.
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ACKNOWLEDGMENTS |
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Received for publication January 23, 2004. Accepted for publication April 15, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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