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Departments of
*
Dermatology and
Medicine, State University of New York, Stony Brook, NY 11794; and
Department of Pathology, Weill College of Medicine, Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin Ab
blocked both adhesion and spreading, anti-
5 integrin
blocked only spreading and not adhesion. Ag capture ELISA of
endothelial cell membrane proteins using polyclonal anti-gC1q-R
showed the presence of not only 1 and 5
integrins but also CD44. Taken together these results suggest that
endothelial cell adhesion and spreading require the cooperation of both
C1qRs and 1 integrins and possibly other
membrane-spanning molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental evidence accumulated from various laboratories including
ours has shown that interaction of endothelial cells with C1q induces a
diversity of functions, including adhesion and spreading
(4); stimulation and expression of the adhesion molecules
E-selectin, ICAM-1, and VCAM-1 (6); and production of
IL-6, IL-8, and monocyte chemoattractant protein-1 (18).
Because C1q is present in high quantities at sites of atherosclerosis
and inflammatory and vascular lesions, and both gC1q-R and
cC1q-R are present on endothelial cells, modulation of endothelial cell
function by soluble and immobilized C1q may contribute significantly to
the development of thrombosis and inflammation. Recent data from our
laboratory (5) have shown that the inflammatory proteins
LPS, TNF-, and IFN-
can up-regulate cell surface expression of
both cC1q-R and gC1q-R on human bone marrow vascular endothelial cells.
Because surface-expressed cC1q-R and gC1q-R have been shown to play a
significant role in a wide range of ligand-mediated cellular responses
(19), we hypothesized that molecules such as cC1q-R and
gC1q-R, which lack direct access to the intracellular space, may
transmit their message by association with other signaling membrane
proteins. The evidence presented in this paper supports our hypothesis
and shows for the first time that C1q-mediated endothelial cell
adhesion and spreading requires the cooperation between C1qRs and
1 integrins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless specified, the following chemicals and reagents were
purchased from the commercial sources indicated: FCS (HyClone
Laboratories, Logan, UT); RPMI 1640, 100x antibiotic-antimycotic
mixture, Dulbecco’s PBS, GRRGDSP and GRRGESP peptides, as well as
anti-5 integrin (P1D6; Life Technologies,
Gaithersburg, MD); and mAb (4B4) to 1 integrin
(Coulter, Hialeah, FL).
Culture of HDMVEC
Human dermal microvascular endothelial cells (HDMVEC) were isolated from human neonatal foreskins as previously reported (20). Briefly, after initial harvest from minced trypsinized human foreskins, microvascular endothelial cells were further purified on a Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient. HDMVEC were cultured on collagen type 1-coated tissue culture flasks in endothelial cell growth medium consisting of endothelial basal medium supplemented with 10 ng/ml epidermal growth factor, 0.4% bovine brain extract, 17.5 µg/ml dibutyryl cAMP, and 1 µg/ml hydrocortisone in the presence of 30% normal human serum. Endothelial cell cultures were characterized and determined to be >99% pure on the basis of formation of typical cobblestone monolayers in culture, positive immunostaining for factor VIII-related Ag (von Willebrand factor), and selective uptake of acetylated low density lipoprotein. All experiments were performed with HDMVEC below passage 10.
Endothelial cell adhesion and spreading assay
The assay used in this study for measuring endothelial cell
adhesion and spreading was modified from a previously described
standard assay (21). Immulon 4 microtiter plates
(Dynatech, Chantilly, VA) were coated with 50 µl/well C1q (10–50
µg/ml) or other purified proteins diluted in 50 mM Tris (pH 7.4), 150
mM NaCl, and 0.1% NaN3. BSA (20 mg/ml) and
heat-inactivated C1q (
C1q; 56°C, 1 h) were used as negative
controls, and type I collagen (10 µg/ml) was used as a positive
control. Plates were incubated overnight at 4°C. All plates were then
washed, incubated with 2% BSA in PBS containing 0.1%
NaN3 for 2 h at room temperature (20°C) to
block nonspecific binding sites, and then washed again before use.
HDMVEC were washed twice, harvested, resuspended in assay buffer
(endothelial basal medium with 0.1% BSA), and incubated with or
without inhibitors for 30 min at room temperature before initiation of
the assay. Cell attachment and spreading were measured by adding
100-µl aliquots of HDMVEC suspension (104
cells/well) to the coated microtiter plate wells and incubating the
plate at 37°C for 1–2 h for attachment and for 6–8 h for spreading.
To assess the potential effect of Abs on cell adhesion or spreading,
the cells were pretreated (30 min, 37°C) with a predetermined
concentration of the anti-C1q-R or anti-integrin Abs before
addition to C1q-coated plates. At the end of the assay 100 µl 2%
glutaraldehyde was carefully added to each well to fix the attached
cells. Then the unattached cells were removed by gently washing the
wells twice with PBS and once with distilled water. Endothelial cell
adhesion and spreading were observed and recorded with a Nikon
Diaphot-TMD inverted microscope (Nikon, Melville, NY) equipped with a
video system consisting of a Dage-MTI CCD-72S video camera and linked
to a Macintosh G3 computer (Apple Computer, Cupertino, CA). The
images were captured at various magnifications using Adobe Photoshop
(Adobe Systems, San Jose, CA). All experiments were repeated at
least three times.
Quantification of cell adherence and spreading
The percentage of adherent cells was calculated in two different
ways. The first involved manual counting under the microscope, the
total number of cells (i.e., spreading and nonspreading (round cells)),
in four randomly selected fields under the microscope by three separate
individuals. The results of each individual count were added and
averaged. The percentage of inhibition was then calculated by
subtracting the number of adherent cells (spreading plus nonspreading)
in each experiment with either mAbs (see Fig. 3
) or polyclonal Ab (pAb;
see Fig. 4) from the control (without Ab) and dividing by 100. In the
second method cell adhesion was quantified spectrophotometrically by
the detergent-compatible bicinchoninic acid method after solubilizing
the cells with 4,4-dicarboxy-2,2'-biquinoline (Pierce, Rockford, IL).
Nonspecific adhesion to BSA- or C1q-coated wells was subtracted
from each experiment. Inhibition of spreading was calculated by
subtracting the number of spreading cells from the total number of
adherent cells in each experiment as described above.
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The confluent HDMVEC monolayers (106/ml)
grown in a collagen-coated flask were surface biotinylated in situ
using the surface-impermeable reagent sulfo-NHS-LC-biotin
(sulfosuccinimidyl-6-biotinamidohexanoate, Pierce) as described
previously (22). Briefly, a culture flask containing 
25
ml HDMVEC was taken out from the incubator, and the excess medium
containing nonadherent cells was discarded. The adherent cells in the
flask were then washed three times in warm HEPES-buffered saline (10 mM
HEPES, 137 mM NaCl, 4 mM KCl, and 11 mM glucose) and incubated (4°C,
2 h) with 10 ml 5 mM sulfo-NHS-LC-biotin. The excess biotin was
removed, and the cells were washed twice in 20 ml HEPES-buffered saline
and lysed using lysis buffer (10 mM HEPES, 150 mM NaCl, 2 mM PMSF, 1
µM aprotinin, 1 µM pepstatin, 1 mM EDTA, 0.1% soybean trypsin
inhibitor, and 1% Nonidet P-40) on ice as described previously
(22). The cell lysate was finally taken out of the flask
and transferred onto sterile test tubes, and after removal of the
nuclei and insoluble cellular debris by centrifugation (15 min,
850 x g, 4°C) the supernatant containing the labeled
proteins was subjected to further centrifugation (1 h, 45,000 x
g, 4°C). The supernatant was then collected, the total
protein concentration estimated by the bicinchoninic acid protein assay
(Pierce), and the degree of biotinylation verified by ELISA using
alkaline phosphatase (AP)-conjugated streptavidin as a probe. The
labeled membrane solution was either used immediately or aliquoted and
kept frozen at -80°C.
Proteins and Abs
Highly purified human C1q was either purified as described
(23) or purchased from Advanced Research Technologies (San
Diego, CA) and was dialyzed against sterile RPMI 1640 or Dulbecco’s
PBS before use in culture with endothelial cells. The production and
characterization of mAb 60.11 and pAb to gC1q-R and to cC1q-R in
rabbits have been described previously, and the IgG fraction was
purified as described previously (24, 25). mAb specific
for 1 integrin (mAb 4B4) was purchased from
Coulter, and anti-5 integrin (mAb P1D6)
was purchased from Life Technologies. The following mAbs, A3D8
(anti-CD44; Mr = 80–110 kDa), AF3
(anti-CD44H; Mr = 100 kDa), P1B5
(anti-3 integrin),
4 (anti-4 subunit
of VLA4), and P1D4 (anti-5 subunit of
integrin), were gifts from Dr. M. Shepley (Division of Infectious
Diseases, State University of New York, Stony Brook, NY).
Electrophoresis and Western blotting
Solubilized membrane proteins from surface-biotinylated HDMVEC
were first prepared as described above, the total protein concentration
was adjusted to 2 mg/ml, and then 100 µg protein was applied to
each lane of a 1.5-mm-thick slab of 10% SDS-PAGE, and the proteins
were separated by electrophoresis under reducing conditions using the
buffer system of Laemmli (26). The separated proteins were
then electrotransferred to polyvinylidene difluoride nitrocellulose
membranes, the membranes were blocked with 5% nonfat milk in TBST (20
mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Tween 20), and the bound
proteins were analyzed by Western blotting using either mAb 60.11 or
nonimmune, species- and isotype-matched IgG. The proteins were then
visualized by sequential reaction with AP-conjugated goat
anti-mouse F(ab')2 and nitroblue tetrazolium
and 5-bromo-4-chloro-3 indolyl phosphate substrate for AP. Detection of
biotinylated proteins was performed using AP-streptavidin, followed by
nitroblue tetrazolium and 5-bromo-4-chloro-3 indolyl phosphate.
AC-ELISA
For the Ag capture ELISA (AC-ELISA), microtiter plates (MaxiSorb; Nunc, Kamstrup, Denmark) were first coated with 100 µl of 10 µg/ml (carbonate buffer, pH 9.5) of either the capturing anti-gC1q-R pAb or nonimmune, species-matched control IgG (2 h, 37°C), washed with TBST, and blocked with 2% BSA. After further washing, 100 µl biotinylated HDMVEC membranes that had been diluted to a concentration of 100 µg/ml in Tris buffer (TB; 20 mM Tris-HCl, pH 7.5) containing 0.5 M NaCl, 0.05% Tween 20, and 0.1% BSA were added to each well and incubated (overnight, 4°C). After incubation, the wells were washed once with TB containing 1 M NaCl and 0.05% Tween 20, twice with TBST, and once with TBS. Washes in high salt are essential to reduce nonspecific binding. The captured proteins were then detected using mAbs to either gC1q-R or integrins and were further developed by standard ELISA. Standard for the capture assay included concentrations (0–1000 ng/ml, in TBS containing 0.5 M NaCl) of highly purified gC1q-R, whereas a similarly treated irrelevant Ag, BSA, was used as control for nonspecificity. To ensure that the captured Ags are surface labeled, a duplicate AC-ELISA was performed under the same conditions, except that the captured Ags were detected by AP-conjugated streptavidin or extravidin and visualized by reaction with p-nitrophenyl phosphate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although other types of endothelial cells (e.g., HUVEC) have been shown previously to express both cC1q-R/CR and gC1q-R/p33 (2, 3, 4), preliminary experiments had nevertheless to be performed to ensure that the HDMVEC used in these studies also expressed these molecules. This was accomplished in two steps. First, solubilized HDMVEC membrane proteins were analyzed by Western blotting using mAb 60.11 anti-gC1q-R and pAb anti-cC1q-R and showed that both molecules are expressed on HDMVEC. Second, surface biotinylated HDMVEC membrane proteins were analyzed by Ag capture assay to determine whether both are labeled specifically. To this end, microtiter wells were coated in quadruplicate with 50 µl (5 µg/ml) each of isotype- and species-matched control IgG, mAb 60.11, pAb anti-cC1q-R, or anti-gC1q-R. After blocking with BSA as described, biotinylated membrane proteins that had been preincubated with mouse Fc fragments to block FcRs were added to the wells and incubated overnight at 4°C. Two of the quadruplicate wells were further reacted with AP-streptavidin and p-nitrophenyl phosphate and showed that the Ags captured by the specific anti-C1q-R Abs were surface biotinylated, whereas no AP-streptavidin-reactive Ags were captured by the wells coated with nonimmune IgG. The protein(s) captured in the other two quadruplicate wells was eluted by the addition of SDS-PAGE sample buffer and incubation for 1 h at 37°C. The eluted proteins from each well were applied to individual lanes of an SDS-PAGE and analyzed by Western blotting using anti-C1q-R Abs and/or AP-streptavidin. The results of these preliminary experiments (data not shown) demonstrated that 1) HDMVEC express both cC1q-R and gC1q-R, and 2) both molecules are surface expressed, as evidenced by biotin incorporation as previously described for Raji cells (22).
Adhesion and spreading of endothelial cells on C1q
Previous studies (3, 6, 12, 13) have shown that
various types of endothelial cells are capable of binding human C1q and
that this binding can trigger biological responses that include
adhesion and spreading (5). Although it is generally
accepted that this binding is mediated via the collagen tail of C1q
(18) and a corresponding cell surface receptor(s), the
identity of the specific receptor(s) involved has not been determined
to date. Using cultured HDMVEC and C1q-coated microplate wells as a
model system we show here that these cells are also able to
specifically bind to and spread on C1q (Fig. 1A). The binding and spreading
were dose dependent and at physiologic ionic strength and did not
require the presence of metal ions. Furthermore, adhesion to C1q was
inhibited when binding was performed in the presence of
(Fab')2 anti-C1q Ab (data not shown) or when
heat-inactivated C1q (56°C, 1 h) was used instead of C1q (Fig. 1B). No adhesion or spreading occurred on wells that had
been coated with 1 mg/ml BSA. The adhesion of HDMVEC to C1q was
quantitatively and qualitatively similar to that of collagen type 1
(Fig. 2).
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To investigate whether the endothelial cell binding is mediated by
cC1q-R and/or gC1q-R, both of which are present on endothelial cells
and implicated in C1q binding (1, 2, 3, 4, 5), HDMVEC were first
preincubated (30 min, 4°C) with concentrations ranging from 10 to 100
µg/ml of either control IgG or mAb 60.11 before addition to
C1q-coated wells. After incubation, the cells were washed, and the
adherent cells were fixed and visualized as described. Fig. 3
shows that spreading (68 ± 12%)
and, to a moderate degree, adhesion (47 ± 9%) were inhibited by
mAb 60.11 in a dose-dependent manner, whereas even the highest dose
(100 µg/ml) of control IgG had no effect. Similar results were
obtained when endothelial cells were preincubated with 30 µg/ml pAb
anti-cC1q-R Ab (Fig. 4). When the two
Abs were mixed in the proportion of 100 µg/ml (60.11) and 30 µg/ml
(pAb anti-cC1q-R), they had a slight (75% ± 13) additive effect
in their ability to inhibit spreading (data not shown).
RGD inhibits C1q-mediated endothelial cell spreading
Using human diploid fibroblasts, it has been shown
previously that adhesion of cells to C1q was inhibited by soluble
GRGDTP peptide (27). This suggested that adhesion of cells
to C1q may require the participation of C1qRs and integrins. To test
this hypothesis, HDMVEC were incubated (30 min, 4°C) with either 30
µM GRRGDSP (RGD) or GRRGESP (RGE) before addition to C1q-coated
wells. After incubation, cells were treated and processed as described
above. As shown in Fig. 5, RGD peptide,
but not RGE, was able to completely (100%) inhibit endothelial cell
spreading, but not adhesion, as assessed by visual examination and
manual counting. Those that remained adherent were round and without
any of the characteristic cytoskeletal reorganization and formation of
typical cobblestone monolayers seen on spreading cells.
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1 integrin on endothelial cell
spreading
To identify the type of integrin(s) that may be involved in
C1q-mediated endothelial cell spreading, AC-ELISA was first performed
on HDMVEC membrane proteins using pAb anti-gC1q-R-coated wells. The
captured proteins were then probed with various anti-integrins as
described in Materials and Methods. The results of this
experiment (Fig. 6) showed that
1 integrin(s) is cocaptured with gC1q-R,
giving strong evidence that 1 integrins and
gC1q-R may collaborate to induce C1q-mediated spreading. In addition,
CD44 was cocaptured by anti-gC1q-R-coated wells.
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1 integrin and
C1qRs cooperate to trigger spreading, HDMVEC were first pretreated (30
min, 4°C) with either 50 µg/ml control IgG or mAb
anti-1 or 5
integrin. The cells (1 x 105/well) were
then added to C1q-coated wells and visually examined for spreading and
adhesion as described above. Fig. 7 is
representative of four such experiments and shows that while mAb
anti-1 integrin inhibited both spreading
and adhesion of HDMVEC to C1q-coated wells,
anti-5 integrin was able to inhibit only
cell spreading. Other experiments (data not shown) similarly designed
to test the effects of other integrins showed that they had minimal or
no significant inhibitory effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address this question, we used an experimental system in which the
adherence and spreading of HDMVEC on C1q-coated microtiter wells was
investigated and the involvement of the two C1qRs in question cC1q-R/CR
and or gC1q-R was assessed. Our results show that HDMVEC, like other
endothelial cell types, can adhere and spread on C1q-coated microtiter
wells in a specific and dose-dependent fashion (Fig. 1), and this
finding is consistent with the long-held postulate that C1q can act as
a matrix protein for endothelial cells and fibroblasts (4, 18, 27). The degree of adherence and spreading was qualitatively and
quantitatively comparable to the adherence of HDMVEC on type I collagen
(Fig. 2). Furthermore, while adherence was not drastically affected,
spreading of HDMVEC to C1q was inhibited when the cells were first
pretreated with either mAb 60.11, an Ab directed to a C1q-binding site
on the N terminus of gC1q-R (Fig. 3), or polyclonal anti-cC1q-R
(Fig. 4) Abs, while species- and isotype-matched control IgG had no
effect on either adherence or spreading. More importantly, however,
spreading was completely inhibited and adherence was moderately
inhibited when HDMVEC were preincubated with 30 µM soluble RGD
peptide, but not RGE, before addition to C1q-coated wells (Fig. 5).
Moreover, pretreatment of HDMVEC with 50 µg/ml mAb
anti-1 integrin before addition to
C1q-coated wells also resulted in the inhibition of adhesion and
spreading, but preincubation of cells with control IgG did not. Of the
anti- integrins tested only anti-5
integrin (Fig. 7) was able to moderately inhibit spreading, indicating
that it may be a likely partner of the 1
integrin. AC-ELISA using anti-gC1q-R also confirmed that
1 integrin is cocaptured from solubilized
HDMVEC membranes (Fig. 6). An unexpected finding was, however, that
both anti-CD44 Abs, A3D8 and AF3, which recognize different
isoforms of the molecule, were positive in AC-ELISA, indicating that
CD44 was also cocaptured by anti-gC1q-R. CD44 is a broadly
expressed, membrane-spanning proteoglycan that binds hyaluronan and is
involved in cell adhesion, signaling, and activation (28).
Alignment of the N-terminal residues 74–161 of gC1q-R/p33 (also known
as p32) with the contiguous transmembrane and cytoplasmic segment of
CD44 (residues 273–360) reveals that 14 aa of 88 possible matches were
conserved and identical (29). Although its role in
C1q-mediated HDMVEC adhesion could not be ruled out by the experiments
in this study, the cocapture of CD44 by anti-gC1q-R pAb may be due
to recognition by the Abs of an epitope(s) in the conserved region
of CD44.
The present studies did not address the relevance in C1q-mediated
adhesion and/or adherence of other C1q-binding molecules such as C1qRp
(7, 30), a homolog of the murine fetal stem cell marker
AA4 Ag (31), which is also expressed on vascular
endothelial cells. Rather, the studies were undertaken to answer a
simple, but intriguing, biological question: how do proteins such as
cC1q-R and gC1q-R, which have been shown to induce biologically
relevant cellular responses, communicate with elements inside the cell
without possessing a membrane-spanning domain? The results presented in
this report collectively suggest that at least on HDMVEC, C1q-mediated
adhesion and spreading may require the participation of cell surface
C1qRs, 1 integrins, and possibly other
molecules. This model would envisage that both cC1q-R and gC1q-R, which
form a high-affinity binding complex upon ligand binding
(25), would laterally associate with
1 integrin to form a signaling complex (Fig. 8). This kind of molecular association
between membrane-spanning and nonmembrane-spanning proteins to form a
docking/signaling complex is not unique to gC1q-R and/or cC1q-R. Many
surface-associated proteins that lack transmembrane domains are known
to trigger biological responses using this strategy. For example, uPAR
(CD87), which is also expressed on vascular endothelial cells, is a
GPI-anchored protein and as such has no direct link with signaling
proteins inside the cell (32). However, uPAR can form a
complex with 1 or 2
integrins to modulate their adhesive functions and with the
2 integrin, CR3 (CD11b/CD18), to trigger
urokinase plasminogen activator-induced Ca2+
fluxes in neutrophils (32, 33). More recently, a signaling
partnership between uPAR and L-selectin (CD62L) was also demonstrated
in human polymorphonuclear neutrophils (34). More
importantly, recent evidence suggests that C1q and mannose-binding
lectin can engage cell surface calreticulin (cC1q-R) and CD91 to
initiate macropinocytosis and uptake of apoptotic cells
(35). Mannose-binding lectin is a member of the family of
proteins collectively known as collectins (collagen containing lectins)
and, like C1q, binds to cC1q-R or CR (or collectin receptor) (9, 36). Apoptotic cell uptake via the CR/CD91 docking/signaling
complex (35) is therefore another example of the molecular
partnership that is forged between transmembrane and nontransmembrane
proteins in the induction of certain biological responses.
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Although both cC1q-R and gC1q-R are constitutively expressed on resting
nonthrombotic endothelium, and gC1q-R in particular has the potential
to activate the bradykinin-generating system (16, 37), the
mechanism by which a continuously thrombogenic state of the endothelium
is averted is not known. We speculate that efficient engagement of
gC1q-R and/or cC1q-R by C1q, C1q-containing immune complexes, or other
ligands is restricted to conditions where under chemical, physical, or
infectious insult, the endothelial cells are converted to a
prothrombotic and proinflammatory phenotype, leading to the induction
of cytokines such as IL-1 or TNF- or the expression of cell adhesion
molecules (6, 18, 39). Leukocytes bound to the cell
adhesion molecules, in turn, can release cytokines, which can amplify
the system by up-regulating the expression of C1q-binding molecules in
a manner that allows efficient binding of ligands such as C1q or HK.
That cytokines such as LPS, IFN-, and TNF- can up-regulate the
expression of both cC1q-R and gC1q-R has been reported previously
(5).
Because C1q is present in high quantities at sites of atherosclerosis and inflammatory and vascular lesions, and gC1q-R as well as cC1q-R and possibly other C1q-binding cell surface proteins are present on endothelial cells, modulation of endothelial cell function by soluble and/or immobilized C1q may therefore contribute significantly to the development of thrombosis and inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Berhane Ghebrehiwet, Department of Medicine, State University of New York, Health Sciences Center, T-16-040, Stony Brook, NY 11794-8161. E-mail address: berhane{at}mail.som.sunysb.edu
3 Abbreviations used in this paper: CR, calreticulin; AC-ELISA, Ag capture ELISA; AP, alkaline phosphatase; C1q, heat-inactivated C1q; HDMVEC, human dermal microvascular endothelial cell; HK, high-m.w. kininogen; pAb, polyclonal Ab; TB, Tris buffer; uPAR, urokinase plasminogen activator receptor.
Received for publication May 16, 2001. Accepted for publication December 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v/3 mRNA by fibrin. J. Invest. Dermatol. 113:913.[Medline]
v and 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen. J. Invest. Dermatol. 106:102.[Medline]
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