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Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison. 6153 Medical Sciences Center, Madison, WI 53706, USA
1Correspondence: University of Wisconsin-Madison, Department of Pathology and Laboratory Medicine, 6153 Medical Sciences Center, 1300 University Ave., Madison, WI 53706, USA. E-mail: afriedl{at}facstaff.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: skin • basement membrane • FGF • cell signaling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
). They signal
through receptor tyrosine kinases (FR) that are encoded by four genes
(FR1-FR4) (reviewed in ref 3
). Their mRNAs undergo
alternative splicing with profound effects on ligand affinity and
specificity.
FGFs are characterized by an affinity for heparin and heparan sulfate
(HS) and in fact, HS is required for the formation of a stable
trimolecular signaling complex with the FGF ligand and FR (4
, 5
; reviewed in ref 6
). The assembly of this
receptor complex is facilitated through HS binding sites on FGF
(7)
and also on FR (8)
. HS may aid in
stabilizing FR dimers or oligomers, allowing transphosphorylation of
the FR cytoplasmic domains and the recruitment of intracellular
signaling proteins (9
10
11
12)
.
There is convincing evidence that different FGFs have distinct HS
requirements with respect to internal HS structure and chain length
(13
, 14)
. Based on different HS binding requirements of
FGF ligands and receptors, HS can also be either stimulators or
inhibitors of FGF signaling. A HS species that has affinity for binding
sites on both FGF ligand and FR will act as a promoter of activity, an
HS that only binds ligand but not FR will be a competitive inhibitor of
activity.
These observations are of profound physiological relevance. Heparan
sulfate proteoglycans (HSPGs) are likely to play an important role in
growth factor regulation. In the developing central nervous system of
mouse embryos, the neuroectodermal cells switch from expressing HSPGs
that favor binding of FGF-2 to a variety that preferentially binds
FGF-1 (15)
. This change occurs at a critical time point
when both growth factors are starting to be produced. In this context,
HS may introduce an additional level of specificity into the signaling
pathway that in the case of FGFs is chararacterized by multiple ligands
potentially interacting with four types of FRs.
HSPGs can be categorized into cell surface and extracellular matrix
forms (reviewed in ref 16
17
18
). The tissue distribution is
determined by the nature of the core protein. Cell surface HSPGs
include the glypican and syndecan families. Perlecan is a secreted HSPG
that is abundantly present in basement membranes. Heparan sulfate
glycosaminoglycan chains are synthesized on the proteoglycan core
protein within the Golgi apparatus. The sugar backbone consists of
repeating disaccharide units, but this initially monotonous chain is
modified by epimerization reactions and the introduction of sulfate
groups in various positions, generating a complex and unique sulfation
pattern (19)
. These modifications generate affinities for
FGFs and other growth factors. Several groups of investigators have
conducted experiments to identify the HS proteoglycan species
responsible for FGF signaling and have reached different conclusions.
Perlecan, syndecan-1, and glypican have all been implicated in FGF
signaling (20
21
22
23
24)
. The explanation for these somewhat
contradictory results might be that cell source and context are more
important determinants of HS chain structure and function than the
identity of the core protein.
These observations illustrate that it would be desirable to investigate
the role of HSPGs in FGF signaling ideally in vivo or
in situ. As a first step in this direction, we have examined
the specificity of FGF-2 and FGF-7 binding to HSPGs in human skin
(25)
. Using biotinylated growth factors as binding probes,
we found that FGF-2 and FGF-7 preferentially bind to distinct HSPGs.
These studies are now being expanded by measuring the ability of tissue HSPGs to promote FGF-2/FR-1 association in situ. This novel method allows us the determination of functional properties of HS regardless of the nature of their core protein in the natural tissue context. Even though basement membrane HSPGs at the dermo-epidermal junction and in dermal micro-vessels bind FGF-2 avidly, these growth factor/HS complexes are incapable of immobilizing soluble FR-1. This result suggests that this class of HSPGs has inhibitory effects on FGF-2 signaling. Conversely, keratinocyte membrane HSPGs bind less FGF-2, but do mediate the assembly of a stable detectable complex with FR-1. Examination of basement membranes in a renal cell carcinoma reveals an identical pattern. These results suggest that extracellular matrix HSPGs such as perlecan can have inhibitory effects on FGF-2 signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. FR1-AP cDNA was cloned into the
pcDNA-3 mammalian expression vector that carries the gentamicin
resistence marker (Invitrogen, Carlsbad, Calif.). The plasmid was
transfected into COS-7 cells with lipofectamin (GibcoLife Technologies,
Inc., Grand Island, N.Y.) using a standard protocol and selection was
carried out with G418 (Geneticin, Life Technologies, Inc.-BRL).
Conditioned medium was collected from the selected polyclonal
population and FR1-AP was purified by affinity chromatography
(anti-AP-agarose, Sigma, St. Louis, Mo.). FR1-AP protein was subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
to determine molecular size and to verify the concentration. FR1-AP and
human placental AP as standard were resolved on 7.5% minigels at
different dilutions with and without prior heating. Total protein was
detected in the gels by staining with SYPRO red (Molecular Probes,
Eugene, Oreg.) and scanning of the stained gels on a Storm
fluoroimager. Alternatively, the proteins were transferred to
nitrocellulose membranes (Bio-Rad, Hercules, Calif.) and analyzed with
anti-AP antibody (Sigma).
FR1-AP binding to cell monolayers
Chinese hamster ovary (CHO) cells were plated at 20,000
cells/well into 96 well plates and fixed after 24 h with 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. The
cell monolayers were incubated with 10 nM recombinant human FGF-2
(provided by B. Olwin, University of Colorado-Boulder) in PBS + 0.1%
bovine serum albumin (BSA) for 1 h at room temperature. After
rinsing, FR1-AP was added at different concentrations for 1 h at
room temperature. Unbound receptor was removed by washing and
immobilized AP activity was determined by adding AP assay mix to the
wells. The 1x assay mix contained: 1M diethanolamine, 0.5 mM
MgCl2, 10 mM homoarginine, 6 mM p-nitrophenyl
phosphate (all from Sigma). Absorbance was measured in a microplate
reader at 405 nM (Bio-Tek Instruments, Winooski, Vt.).
In situ assays for FGF and soluble receptor binding
Human tissue was obtained fresh from the operating room,
embedded in OCT compound, snap frozen and stored at -70°C. NIH
guidelines for the use of human material were followed and
institutional review board approval was obtained. Binding assays were
carried out essentially as described previously (25). Five µm thick
cryostat sections were thaw-mounted on charged slides (Fisher ‘plus’
slides), immediately dipped in 95% ethanol, and fixed in 4%
paraformaldehyde in PBS at room temperature. The fixation procedure is
crucial for reproducible FR1-AP binding of high intensity. After
blocking with BSA (10 mg/ml) in PBS, the sections were incubated with
FGF-2 (10 nM) for 60 min. at room temperature. To detect growth factor
binding, monoclonal anti-FGF-2 antibody (DE-6, provided by Dupont,
Wilmington, Del.) was added for 1 h at room temperature. To
determine soluble receptor binding, FR1-AP was added at varying
concentrations (30 nM for most experiments including the images shown).
Immobilized soluble receptor was detected with monoclonal anti-AP
antibody (Sigma). Controls for soluble receptor binding included
omission of the FGF-2 incubation step and digestion with heparan
sulfate lyases prior to FGF-2 exposure (see below). Both primary
antibodies (anti-FGF-2 and anti-AP) were visualized with TRITC or
Alexa-546-conjugated donkey anti-mouse antiserum (Jackson
ImmunoResearch, West Grove, Pa.; and Molecular Probes, respectively).
The slides were then rinsed and coverslips were mounted with Immumount
(Shandon, Pittsburgh, Pa.).
Heparitinase digestion
Tissue sections were exposed to heparitinase enzymes to identify
the FGF-2 binding sites as HS and to generate epitopes recognized by
the anti-heparan sulfate antibody clone 3G10 (see below). Heparitinase
(mixture of 95% heparitinase I and 5% heparitinase II, Seikagaku,
Ijamsville, Md.) and pure heparitinase II (ICN, Costa Mesa, Calif.)
were reconstituted and diluted in heparitinase buffer (50 mM Hepes, 50
mM NaOAc, 150 mM NaCl, 9 mM CaCl2, 0.1% BSA, pH
6.5). The enzymes were used at a final concentration of 4 mIU/ml either
alone or in combination (as indicated) in the Sequenza racks at 37°C
for varying amounts of time. When complete digestion was intended, the
slides were incubated for 4 h, replacing the enzyme after 2 h.
Immunolabeling of heparan sulfate
Total HS was detected in tissue sections with monoclonal anti-HS
antibodies. Antibody 3G10 (Seikagaku) detects desaturated uronic acid
residues that remain after digestion of HS with heparitinase
(27)
. This epitope specificity allows the detection of HS
stubs that remain after heparitinase digestion regardless of the
original structure or sulfation pattern of the HS chain. The antibody
was used at a dilution of 1:200 (5 µg per ml).
Slide viewing and image analysis
Sections were examined using a Nikon Microphot FX microscope
equipped for epifluorescence. Images were acquired with a Photometrics
CCD camera (Tucson, Ariz.) and Image-Pro-Plus analysis software (Media
Cybernetics, Silver Spring, Md.). In some experiments, density
measurements were carried out using NIH Image 1.62 software on an Apple
Power PC computer. For this purpose, all images in one data group were
acquired at the same parameters (exposure time, gain) and saved as
uncompressed TIFF (Tagged-Image File Format) images. Signal intensity
measurements were carried out using the line plot profile tool in NIH
image by drawing a line perpendicular across the structure of interest
(keratinocyte membrane or epidermal basement membrane). The
maximal signal for each line was measured and exported into
a spreadsheet program (Microsoft Excel) for statistical analysis.
Approximately 25 measurements were performed per image, with 2 or 3
images per slide.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A weaker band of ~160 kDa likely represents a degradation product
rather than FR1-AP monomer. Under the conditions used, SDS denaturation
does not dissociate the dimers without heating. It is likely that the
dimer formation is mediated by the AP portion of the molecule. Since
human placental AP enzyme is functional only as a homodimer
(28)
, it is apparent, that only dimers are detected by
enzyme activity assays. This includes experiments measuring FR1-AP
binding to cell monolayers where binding is quantified via AP activity.
This is fortunate because it allows the simulation of receptor
dimerization in solution which physiologically occurs in two dimensions
within the cell membrane. The existence of soluble receptors as dimers
probably accounts for the relatively high affinity of FR1-AP for FGF-2
in solution (26)
. Dimerized soluble receptor therefore can
be used as a binding probe in situ to serve as a surrogate
marker for signaling receptor assembly.
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FR1-AP-soluble receptor binding to cell monolayers in
vitro
FR1-AP binding to tissue HSPGs was simulated in vitro
using fixed monolayers of CHO cells. This approach allows easy
quantitative assessment of bound soluble receptor using the AP enzyme
activity. In addition, the effect of HSPGs can be assessed by comparing
wild-type CHO cells with HS-deficient mutants (29
, 30)
.
Binding of FR1-AP to the cell monolayers is highly dependent on the
presence of functional HSPGs and on the presence of prebound FGF-2
(Fig. 2
). No binding to HSPGs alone is detected despite the presence of an HS
binding site on the receptor. FR1-AP binding is also abrogated by
treating wild-type CHO cells with heparitinase (data not shown). These
in vitro observations on cell monolayers are a successful
proof of principle for using FR1-AP as a binding probe on tissue
sections.
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FGF-2 and FR1-AP-soluble receptor binding in situ
The goal of these experiments was to map in intact human tissues
HSPGs that are capable of supporting stable trimolecular receptor
complex formation. This approach is based on the hypothesis that a
specific pattern of modifications (sulfation and epimerization) and
spacing of modified regions are required for proper binding of the HS
chain to both FGF ligand and to the receptor. Conversely, HS species
that bind FGF but not the FR are likely to act as competitive
inhibitors of signaling. This assay is novel because it allows
correlation of tissue structure and molecular function. Skin was chosen
as a model because we have shown previously that FGFs bind to distinct
HS classes in this organ (25)
and because FGFs play an
important role in skin maintenance and wound healing (31)
.
Bound soluble receptor or FGF-2 ligand were detected by
immunofluorescence (Fig. 3
). As reported previously in skin (25)
, FGF-2 localizes to
keratinocyte surfaces, but the strongest binding occurs on basement
membranes both at the dermo-epidermal junction and in the dermal
microvasculature (Fig. 3D
). The pattern of soluble receptor
binding (Fig. 3A
) is distinctly different from that of FGF-2
binding. FR1-AP binds primarily to HSPGs on keratinocyte surfaces in
the epidermis. In stark contrast to FGF-2 binding, no signal is
detected on epidermal or dermal microvascular basement membranes,
despite the fact that FGF-2 at a concentration of 10 nM preferentially
localizes to these structures. Apparently, only a subset of
FGF-2-binding HSPGs promotes receptor binding. Soluble receptor binding
is restricted to cell surface HSPGs (presumably syndecans and
glypicans, while it is lacking in the secreted basement membrane HSPGs
that are likely predominantly perlecan and its variants. The binding
experiments were repeated at least five times using skin samples of
three individuals with identical results.
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The binding of FR1-AP-soluble receptor is highly dependent on the
presence of immobilized exogenous FGF-2, since omission of the growth
factor incubation step completely abrogates binding (Fig. 3B
). The identity of the tissue binding sites is confirmed
as HS by heparitinase treatment, which eliminates FR1-AP binding signal
(Fig. 3C
). Furthermore, visualization of total tissue HS
with monoclonal antibody 3G10, which decorates epitopes generated by
heparitinase treatment, reveals a tissue distribution pattern identical
to that seen by FGF-2 binding (Fig. 3E
). Apparently, FGF-2
is rather nonselective in binding to tissue HSPGs. Without heparitinase
exposure, antibody 3G10 does not label heparan sulfate (Fig. 3F
).
Next, we decided to determine, whether the inability of basement
membrane HS proteoglycans to promote signaling complex assembly was
limited to skin or was a more generalized phenomenon. For this purpose,
tissue from papillary (chromophilic) renal cell carcinomas was
examined. This rather rare kidney neoplasm produces ample basement
membrane material. Consistent with the findings in skin, no binding of
soluble FR1-AP receptor to basement membrane material is detected
(Fig. 4A
). Conversely, the basement membrane that lines the stromal
papillary stalks of the tumor avidly binds FGF-2 (Fig. 4B
).
As in skin, the location of FGF-2 tissue binding sites codistributes
with total HS proteoglycan detected with 3G10 antibody Fig. 4C
). As in skin, FGF-2 (Fig. 4D
) and FR1-AP (not
shown) binding is eliminated by heparitinase treatment.
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Image analysis
The differential ability of tissue HSPGs to promote soluble
receptor binding is likely either due to differences in internal
structure or chain length of the HS molecules. Alternatively, it is
possible that the high density of HS in the basement membrane and
subsequently the high local concentration of FGF-2 leads to steric
hindrance of binding. An analogous scenario is observed in
vitro, where increasing concentrations of heparin, a specialized
mast cell HS, result in a bi-phasic FGF-2 binding pattern to
cell-associated FR with virtual elimination of recognizable binding at
heparin concentrations of 10 µg/ml (32)
. If this
phenomenon were responsible for lack of FR1-AP binding to basement
membrane HSPGs, it would be expected that a gradual reduction in the
number of HS binding sites would generate a transient affinity for
FR1-AP-soluble receptor in the basement membranes.
To test this hypothesis, tissue sections were digested with heparan
sulfate lyases for different time periods and FR1-AP, FGF-2, and 3G10
binding were monitored. The fluorescence intensities in the epidermal
basement membrane and on keratinocyte surfaces were qualitatively
assessed by image analysis using NIH Image software by measuring
maximal fluorescence intensity using the line profile tool
(schematically displayed in Fig. 5A
). Signal intensity of FR1-AP binding to FGF-2 on epidermal
basement membrane is very low prior to enzyme exposure and is further
reduced by sequential digestion with heparitinase (Fig. 5D
,
filled circles). At no point during the digestion is an affinity for
FR1-AP generated. FR1-AP binding to FGF-2 at keratinocyte surfaces
starts out with a higher signal that is rapidly eliminated by
heparitinase (Fig. 5D
, open circles, Fig. 5F, H
).
FGF-2 binding to epidermal basement membrane and to a lesser degree to
keratinocyte surfaces is initially (after 15 min) significantly
augmented by heparitinase treatment (Fig. 5C, E
). At this
time point FR1-AP binding to basement membrane HS is actually reduced
compared to the starting point (Fig. 5D, F
), demonstrating
that in the absence of correct presentation of FGF-2 by the HS stable
receptor binding cannot be achieved. This increase in FGF-2-binding
early during enzyme digestion is probably due to better accessability
of the highly sulfated HS regions by FGF-2 after preferential digestion
of the less sulfated stretches by the enzyme. Eventually, FGF-2 binding
is reduced below the baseline (Fig. 5C, G
). The final
reduction of FGF-2 binding to keratinocyte surfaces is less pronounced
and it can not entirely be excluded that FGF-2 binds also to sites
other than HS. The progress of heparitinase action was also monitored
by determining the abundance of desulfated uronate epitopes recognized
by antibody 3G10. This method shows a nearly linear increase in signal
intensity over the first 60 min (Fig. 5B
). In summary, these
results demonstrate that steric hindrance is an unlikely explanation
for absence of FR1-AP-soluble receptor binding to basement membrane
HSPGs but that rather structural differences in HS chain composition
are responsible.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This unexpected result raises interesting questions in terms of its
biological relevance. FGF-2 appears to play important roles in
maintaining skin integrity. Recent data using FGF-2 knock-out mice
(31)
show that delayed skin wound healing was one of the
few observed phenotypic abnormalities. The epidermal basement membrane
may act as a reservoir for FGF-2 where it is maintained in an inactive
state by HSPGs and is ready to be released after injury. Similarly, it
has been a mystery why FGF-2, one of the most potent angiogenic factors
could be accumulated in the basement membrane of blood vessels in
immediate contact with endothelial cells without inducing mitogenesis
in this cell type (33)
. Indeed, endothelial cells have a
very low turn-over rate in the resting state. The lack of correlation
between the amount of (basement membrane-associated) FGF-2 in tissues
and angiogenic activity has been used to discredit the importance of
FGF-2 as angiogenesis factor (34)
. This apparent paradox
is readily explained by our observation that vascular basement membrane
HSPGs immobilize FGF-2 with high capacity but fail to mediate complex
formation with FR1. Recent experimental evidence exploring the kinetics
of FGF/HS interactions with biosensor devices convincingly supports our
observations. Fernig and co-workers determined association and
dissociation rates of FGF-1 and FGF-2 on immobilized HS isolated from
different mammary gland cell lines (35)
. Myoepithelial
cells that are responsible for basal lamina production in the mammary
gland, secreted HSPGs with a biphasic kinetic profile characterized by
slow association and low capacity and by fast association and high
capacity respectively. These HSPGs were not capable of facilitating
FGF-2 signaling in HS deficient fibroblasts.
The molecular basis for selective HS/protein interactions and
activating vs. inhibitory activity of HS chains is now emerging. HS
chains with a high affinity for FGF-2 are characterized by longer
(e.g., five to six disaccharide units) stretches of N-sulfated
glucosamine and 2-O-sulfated iduronate (36)
. These regions
alternate with areas of less sulfated and N-acetylated saccharides. The
presence of 6-O-sulfates does not appear to increase the affinity of HS
for FGF-2. The 6-O-sulfate moiety is, however, important for binding of
the HS/FGF-2 complex to the FR receptor tyrosine kinase
(13)
. Recent cloning of the enzymes responsible for HS
sulfation has shown that 2-O sulfation and 6-O sulfation are catalyzed
by two different gene products allowing differential regulation of
their activity (37
, 38)
. Dissociation of 2-O and 6-O
sulfation has been observed in several physiological and pathological
scenarios. Maturation of mouse neuroepithelial cells is accompanied by
an increase in 6-O sulfation (39)
. Conversely, in
vitro progression of colon adenoma cells to carcinoma cells and
malignant transformation of mouse mammary tumor cells are associated
with a decrease in 6-O sulfated HSPGs (40
, 41)
.
Investigating the regulation of HS synthesis and modification remains a
challenging task. The role of sulfotransferases has already been
mentioned. In addition, enzymes conducting epimerization and
N-deacetylation/sulfation are involved in the process of HS maturation
(17
, 42)
. Further processing of mature HS chains can also
modulate their activity after the molecules have been inserted into the
cell membrane or have been secreted. The transmembrane HS proteoglycan
syndecan-1 can be shed into wound fluid by proteolysis
(43)
. The released syndecan-1 extracellular domain acts as
an inhibitor of FGF-2 activity. Subsequent digestion of poorly sulfated
regions within its HS chain by platelet-derived heparitinases results
in conversion of the molecule into a potent activator of FGF signaling
(44)
. We are observing a transient increase in FGF-2
binding on keratinocyte surfaces and in the epidermal basement membrane
while the tissue sections are treated with (bacterial) heparitinase
that is not accompanied by increased soluble receptor binding. This
effect is also likely due to degradation of less sulfated HS stretches
and resulting improved accessability of the remaining highly
sulfated/modified HS regions. The effect of heparitinase digestion on
vascular BM is unknown but it has been shown that heparan sulfate
lyases and proteases in cancer and inflammation can release FGF from
its basement membrane storage site.
The question of which HS proteoglycan species mediate FGF signaling has
been the subject of a considerable debate. Yayon has identified the
extracellular matrix HS proteoglycan perlecan as the only one isolated
from human lung fibroblasts with FGF activating ability, while
syndecans and glypican were inactive or inhibitory (20)
.
Nurcombe and co-workers also extracted a perlecan variant as the HS
proteoglycan mediating FGF binding in developing mouse neuroepithelium
(45)
. Conversely, Jalkanen, David and Rapraeger determined
that syndecan-1 not only binds FGF-2, but also mediates FR binding and
activation (21
, 22)
. It has been shown that glypican-1 is
responsible for modulating FGF-1 and FGF-7 effects on keratinocytes
in vitro (23)
. These apparently contradictory
observations can be reconciled with the notion that cell source, tissue
context and metabolic state (e.g., resting vs. reparative) are more
important determinants of HS chain composition than the nature of the
core protein. Bernfield’s recent observations that enzymatic activity
can convert an inhibitor into an activator of FGF support this
hypothesis (44)
. It becomes apparent that novel
experimental approaches such as in vivo and in
situ assay systems are required to investigate the role of HSPGs
as modulators of growth factors in a physiologically relevant
manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. Melrose, S. Smith, P. Ghosh, and J. Whitelock Perlecan, the Multidomain Heparan Sulfate Proteoglycan of Basement Membranes, Is also a Prominent Component of the Cartilaginous Primordia in the Developing Human Fetal Spine J. Histochem. Cytochem., October 1, 2003; 51(10): 1331 - 1341. [Abstract] [Full Text] [PDF]
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S. J. Goodman, C. S. Branda, M. K. Robinson, R. D. Burdine, and M. J. Stern Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity Development, August 15, 2003; 130(16): 3757 - 3766. [Abstract] [Full Text] [PDF]
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D. Qiao, K. Meyer, C. Mundhenke, S. A. Drew, and A. Friedl Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Signaling in Brain Endothelial Cells. SPECIFIC ROLE FOR GLYPICAN-1 IN GLIOMA ANGIOGENESIS J. Biol. Chem., April 25, 2003; 278(18): 16045 - 16053. [Abstract] [Full Text] [PDF] |
