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J Biol Chem, Vol. 274, Issue 44, 31740-31749, October 29, 1999
From the Recent studies suggested that interruption of the
interaction of advanced glycation end products (AGEs), with the
signal-transducing receptor receptor for AGE (RAGE), by administration
of the soluble, extracellular ligand-binding domain of RAGE, reversed
vascular hyperpermeability and suppressed accelerated atherosclerosis
in diabetic rodents. Since the precise molecular target of soluble RAGE
in those settings was not elucidated, we tested the hypothesis that
predominant specific AGEs within the tissues in disorders such as
diabetes and renal failure,
N Receptor for AGE1
(RAGE), a member of the immunoglobulin superfamily, was first described
as a cell surface interaction site for advanced glycation end products
(AGEs), products of glycation and oxidation of proteins and lipids
(1-2). AGEs are a heterogeneous class of compounds, whose accumulation
in disorders such as diabetes, renal failure, Alzheimer's disease,
and, indeed, natural aging, albeit to a lesser degree, has suggested
their potential contribution to the pathogenesis of complications that
typify these conditions (3-7). Our previous studies demonstrated that
both in vitro and in vivo derived heterogeneous
AGEs ligate cell surface RAGE on endothelium (ECs), mononuclear
phagocytes (MPs), vascular smooth muscle (VSMC), and neurons to
activate cell signaling pathways such as ERK1/ERK2 kinases and NF- Our recent studies suggested that interruption of the interaction of
AGEs with RAGE in vivo, by administration of soluble RAGE
(sRAGE), the extracellular ligand-binding domain of RAGE, reversed
vascular hyperpermeability and suppressed accelerated atherosclerotic
lesion development and complexity in diabetic rodents (19-20). In the
latter studies, analysis of plasma demonstrated evidence of an
sRAGE·AGE complex; immunoprecipitation of plasma obtained from
diabetic sRAGE-treated mice with anti-RAGE IgG yielded species
immunoreactive with both anti-RAGE IgG or affinity purified anti-AGE
IgG, suggesting that sRAGE might bind up AGEs and limit their
interaction with and activation of cell surface RAGE. The beneficial
effects of sRAGE were independent of alterations in other risk factors,
such as hyperglycemia and hyperlipidemia, implicating a role for
AGE-RAGE interaction in the development of vascular dysfunction in
diabetes (20).
These past studies, however, did not elucidate the precise AGE(s) that
trigger signal transduction mechanisms upon engagement of RAGE. We thus
sought to test specific AGE structures for their ability to bind RAGE
on the surface of cells such as ECs, MPs, and VSMCs in order to
determine their role in cellular activation.
In this context, recent biochemical and immunohistochemical studies
suggested that N Furthermore, recent findings suggested that CML modifications may form
directly as a consequence of activation of the myeloperoxidase-hydrogen peroxide-chloride system, thereby providing a mechanism for conversion of hydroxy amino acids into glycoaldehyde, a precursor in the steps
leading to formation of CML (34). These findings may have direct
implications for inflammatory processes that characterize certain
complications of diabetes and renal failure, for example, such as
atherosclerosis, impaired wound healing, aggressive and inflammatory
periodontal disease, and dialysis-related amyloidosis (DRA) (6,
35-36).
Consistent with these concepts, previous studies placed RAGE in the
vascular and inflammatory milieu characteristic of disorders in which
AGEs, such as CML, accumulate; indeed, expression of RAGE is enhanced
in these settings, beyond that observed in normal adult tissues
(37-41). Taken together, therefore, these considerations suggested
that examination of CML adducts of proteins as potential specific AGE
ligands for RAGE was a logical step.
Here we report that CML-modified proteins engage cellular RAGE in
vitro and in vivo to activate key cell signaling
pathways such as the transcription factor NF- Synthesis and Characterization of CML-modified
Proteins--
Keyhole limpet hemocyanin (KLH), bovine serum albumin
(BSA), or ovalbumin (OVA) (Sigma) (176 mg in each case), and sodium cyanoborohydride (28.3 mg; 0.45 M) were dissolved in sodium
phosphate buffer (0.2 M; pH 7.8); to this was added
glyoxylic acid (14.3 mg; 0.155 M) in a total volume of 1 ml
per reaction. The mixture was incubated for 24 h at 37 °C.
Control proteins were prepared under the same conditions, except that
glyoxylic acid was omitted (23). These published methods were modified
in order to prepare proteins with a range of CML modifications as
follows: protein was incubated in buffer exactly as above, except that
varying amounts of glyoxylic acid were added as follows: 10 mg (0.108 M), 7 mg (0.076 M), 4 mg (0.043 M),
2 mg (0.0217 M), and 1 mg (0.0108 M);
concomitantly varying amounts of sodium cyanoborohydride were added as
follows: 19.8 mg (0.3152 M), 14.1 mg (0.225 M), 7.1 mg (0.1125 M), 3.5 mg (0.0556 M), and 2 mg
(0.0318 M), respectively, as described previously.
Preparations of CML-modified proteins were extensively dialyzed
versus phosphate-buffered saline (PBS) and characterized by
percent modification as determined both by employment of
2,4,6-trinitrobenzene sulfonic acid to determine the difference in
lysine residues of modified versus unmodified preparations
(42) and by gas chromatography-mass spectroscopy (43). To test
CML-modified adducts, 0.8 mmol of CML/mol of Lys, CML-OVA, 33 mmol of
CML/mol of Lys, were diluted 1:40 with native OVA in PBS. All
CML-modified native proteins, antibodies/F(ab')2 fragments,
and control proteins were devoid of endotoxin prior to experiments by
chromatography onto Detox-igel columns (Pierce). The level of endotoxin
in all protein preparations (concentration range, 2-6 mg/ml) was less
than 3 pg/ml (Sigma).
Preparation and Characterization of Antibodies--
Anti-human
RAGE IgG and affinity purified anti-CML IgG were prepared and
characterized as described previously (44 and 24, respectively). In the
latter case, CML-BSA and CML-KLH prepared as above were employed to
immunize New Zealand White rabbits (24). After 6-12 weeks, serum was
obtained and IgG prepared (Pierce). Affinity purification of the IgG
fractions was performed as follows: IgG fractions were chromatographed
onto Affi-Gel 15 resin (Bio-Rad), which had previously adsorbed KLH.
Material that did not adhere to the resin was collected and
subsequently chromatographed onto Affi-Gel 15 (Bio-Rad) which had
previously been adsorbed CML-BSA. After extensive washing in
Tris-buffered saline (Tris, 0.02 M; pH 7.4, and NaCl, 0.1 M) containing Tween 20 (0.05%), fractions were eluted in
buffer containing glycine, 0.02 M; pH 2.5. A280 nm of each fraction was determined;
positive fractions were immediately neutralized and dialyzed
versus Tris-buffered saline. Enzyme-linked immunosorbent
assays (ELISA) were performed to characterize the affinity purified
antibodies. To wells of plastic dishes (Maxisorp, Nunc, Naperville, IL)
was added 5 µg of the following proteins in bicarbonate/carbonate
buffer, pH 9.6: BSA, KLH, CML-BSA, or CML-KLH. After incubation for
16 h at 4 °C, wells were washed in PBS and then unoccupied
sites on the wells blocked in the presence of PBS containing BSA (1%)
and goat serum (5%; Sigma) for 1 h at 25 °C. Wells were washed
in PBS containing Tween 20 (0.05%) and primary antibody, and affinity
purified anti-CML IgG was added at a concentration of 3 µg/ml in
blocking buffer as above for 3 h at 25 °C. Wells were washed
five times in PBS, and then sites of primary antibody binding were
identified upon incubation with blocking buffer containing
peroxidase-conjugated goat anti-rabbit IgG according to the
manufacturer's instructions (Sigma) for 1 h at 25 °C. Wells
were washed five times and developed in citrate buffer containing
o-phenylenediamine/H2O2 as
instructed (Sigma). In these assays, only CML modifications of proteins
were highly reactive; no reactivity was identified versus
native, unmodified proteins. Furthermore, upon incubation of affinity
purified anti-CML IgG with excess CML-BSA, immunoreactivity of
CML-modified adducts was abolished.
Isolation of Human Serum Albumin and Immunoblotting--
Plasma
samples were obtained from human donors with diabetes, renal failure,
or age-matched healthy controls in accordance with the procedures of
the Institutional Review Board of Columbia University. Samples (2 ml)
were dialyzed for 16 h at 4 °C in phosphate buffer, 0.02 M; pH 7.1 (total volume, 4,000 ml). After dialysis, samples
were subjected to filtration, 0.8 µm, and then chromatographed onto
columns containing Affi-Gel blue resin (Bio-Rad) previously equilibrated in phosphate buffer, 0.02 M; pH 7.1; 5 ml of
resin were employed per ml of plasma. The resin was washed in 2.5 column volumes of phosphate buffer as above, and human serum albumin was eluted by application of phosphate buffer containing NaCl (1.4 M). The A280 nm for each fraction
was determined (Ultraspec Plus, Amersham Pharmacia Biotech), and
positive fractions were determined by SDS-PAGE followed by staining
with silver (Bio-Rad). Immunoblotting was performed employing the above
material; to each lane of SDS-PAGE gels (8%), 30 µg of protein was
added. Simultaneously, marker proteins were added as a means to assess
Synthesis of Advanced Glycation End Products (AGEs) and
Determination of Components That Bind RAGE--
Human IgG (Sigma), 5 mg/ml in PBS, was subjected to nonenzymatic glycation by incubation in
PBS containing D-ribose, 0.025 M (Sigma). The
solution was sterile-filtered (0.2 µm) and then incubated at 37 °C
for 6 weeks under aerobic conditions. At the end of that time, the
mixture was extensively dialyzed versus PBS at 4 °C to
remove unreacted ribose. This material was then chromatographed onto
resin containing Affi-Gel 10 (Bio-Rad) to which had previously been
adsorbed recombinant human soluble RAGE. After incubation, the resin
was washed extensively with 10-column volumes of Tris-buffered saline
(Tris, 0.02 M, pH 7.4; NaCl, 0.1 M) and elution
of bound components performed employing glycine, 0.02 M; pH
2.5. The A280 nm of each fraction was
determined; positive fractions were immediately neutralized and
dialyzed versus TBS. ELISA was performed essentially as
described above employing varying concentrations of CML-BSA as standard
reference. Wells of plastic dishes were coated with AGE-IgG, material
eluted from the above column, and volume control (buffer retrieved
after elution of RAGE-binding components subsequent to washing with
10-column volumes of glycine buffer). Primary antibody, affinity
purified anti-CML IgG was employed (3 µg/ml) to identify
CML-immunoreactivity in the various fractions. Finally, goat
anti-rabbit IgG was added to detect sites of binding of primary
antibody, and plates were developed as above.
A490 nm was detected.
Radioligand Binding Assays--
Human soluble recombinant RAGE
was radiolabeled with 125I by incubation with IODO-BEADS
(Pierce) to a specific activity of Assessment of Cell Surface VCAM-1 and Binding of Radiolabeled
Molt-4 Cells to Stimulated Human Umbilical Vein Endothelial Cells
(HUVEC)--
Human umbilical vein endothelial cells (HUVECs) were
isolated and characterized as described (46). Cells were cultured in 96-well tissue culture-treated wells (Corning, Corning, NY) in endothelial cell growth medium (Clonetics, San Diego, CA) until achieving confluence; medium was then changed to F-12 without serum
(Life Sciences) immediately prior to stimulation with the indicated
concentrations of CML-ovalbumin or native ovalbumin. Where indicated,
cells were pretreated with rabbit anti-human RAGE IgG, nonimmune rabbit
IgG for 2 h; in certain cases, CML-OVA was pretreated with the
indicated concentration of sRAGE for 1 h prior to cell
stimulation. After 6 h, cells were fixed in medium containing
paraformaldehyde (2%) for 10 min followed by incubation in fresh
paraformaldehyde (2%) for 16 h at 4 °C. Wells were washed twice with PBS and incubated with H2O2 (0.3%)
for 10 min at room temperature. Wells were again washed twice with PBS
and incubated in PBS containing BSA (2%) and non-fat dry milk (4%) to
block nonspecific binding sites on the cell surface for 30 min at room temperature. After washing once in PBS, cell surface ELISA employing anti-VCAM-1 IgG (Santa Cruz Biotechnologies, Santa Cruz, CA) (2 µg/ml) was performed for 2 h at 37 °C. Wells were washed four times and incubated with peroxidase-conjugated rabbit anti-goat IgG
(Sigma) for 1 h at 37 °C. Wells were washed five times and sites of antibody binding detected with OPD as above; measurements of
A490 nm per well were obtained. Assessment of
functional VCAM-1 activity was determined using
51Cr-labeled Molt-4 cells (ATCC) as described
(14).
Chemotaxis Assays--
Chemotaxis assays were performed as
described (10) in 48-well microchemotaxis chambers (Neuro-Probe,
Bethesda, MD) containing a polycarbonate membrane (8 µm; Nucleopore,
Pleasanton, CA). Molt-4 cells, which bear cell surface RAGE, were grown
in suspension in medium containing RPMI 1640, fetal bovine serum
(10%), and antibiotics (Life Sciences). The lower chamber contained
the chemotactic stimulus as indicated. N-Formyl-Met-Leu-Phe
(Sigma) was employed as positive control. Molt-4 cells were added to
the upper chamber (5 × 104 cells/well). After
incubation for 4 h at 37 °C, nonmigrating cells on the upper
surface of the membrane were gently scraped and removed; the membrane
was then fixed in methanol (100%), and cells that had migrated through
the membrane were stained with Giemsa (Sigma). Cells in nine
high-powered fields were counted and mean ± S.E. reported. Where
indicated, cells were incubated with the indicated F(ab')2
for 2 h at 37 °C prior to assay; in certain cases, CML-modified
proteins were incubated with the indicated molar excesses of human
soluble RAGE for 1 h at 37 °C prior to assay. In other
experiments, cells were transfected with a construct encoding human
RAGE in which the cytosolic domain (tail) was deleted employing
superfect (Qiagen, Valencia, CA) (2 µg DNA/ml medium); pcDNA3
(Invitrogen) was employed as vector. Stimulation experiments were
performed 48 h after transfection. In cells transfected with RAGE-tail deletion construct, the ability to bind ligand is retained as
RAGE-lacking cytosolic domain is firmly anchored in the membrane (41).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
HUVEC, murine macrophage BV-2 cells (47), and
rat vascular smooth muscle cells (VSMC; generously provided by Dr.
Abraham Rothman) (9) were incubated with the indicated CML-modified or
native protein for 6 h at 37 °C in the presence or absence of
preincubation with the indicated IgG or sRAGE/control protein for 2 and
1 h, respectively, at 37 °C. In certain cases, transient transfection was performed employing mock, or RAGE tail deletion, constructs as above, except that DNA, 0.4 µg/ml medium, was employed. Nuclear extracts were prepared (48) and EMSA performed employing 32P-labeled probe for NF- In Vivo Studies: Infusion of CML-modified and Native
Proteins--
BALB/c mice (Charles River), approximately 6 weeks of
age, were injected intravenously via the tail vein with CML-modified native protein or lipopolysaccharide. Where indicated, animals were
pretreated with the indicated IgG 24 h prior to and at the time of
infusion of CML-modified proteins. In other cases, CML-modified protein
was incubated with excess sRAGE for 1 h prior to infusion. Twelve
h later, lungs were rapidly harvested. Lung tissue was homogenized in
Tris-buffered saline containing protease inhibitor (Roche Molecular
Biochemicals) and subjected to centrifugation at 8,000 rpm for 10 min.
Supernatant was centrifuged for 1 h at 4 °C at 40,000 rpm and
the pellet dissolved in TBS containing protease inhibitors and octyl
Statistical Analysis--
Statistical comparisons were
determined using one-way analysis of variance; where indicated,
individual comparisons were performed using Student's t
test. In all cases, statistical significance was ascribed to the data
when p < 0.05.
CML Modifications Are Present to Enhanced Degrees in Human Serum
Albumin in Diabetes and Renal Failure and Bind RAGE
As a first step in elucidating whether CML modifications occurred
in typical proteins such as albumin, we isolated human serum albumin
from age-matched subjects with diabetes, renal failure, or controls by
chromatography of plasma on Affi-Gel blue. Albumin preparations,
analyzed by SDS-PAGE and immunoblotting with affinity purified anti-CML
IgG, displayed Thus, CML-BSA was prepared and tested for its ability to bind RAGE.
When immobilized onto the wells of plastic dishes, CML-BSA bound
125I-sRAGE in a dose-dependent manner, with
Kd Further evidence for selectivity of binding was sought by identifying
structural determinants within RAGE that mediated interaction with
CML-BSA. Three distinct RAGE domain-specific fusion proteins were
prepared with glutathione S-transferase (GST) by expression in E. coli followed by thrombin treatment to remove GST.
RAGE domains were then purified to homogeneity. By amino-terminal amino acid sequencing and SDS-PAGE, purified V domain, C1 domain, and C2
domain (1-2) were obtained (data not shown). By using purified RAGE
domains, competitive binding studies were performed with 125I-sRAGE and immobilized CML-BSA; addition of a 50-fold
molar excess of unlabeled V domain significantly suppressed binding,
whereas C1 and C2 domains were without effect (Fig. 1D).
These data implicated the V domain of RAGE in specific binding to
CML-modified adducts.
Multiple studies have indicated that a dominant species in the
heterogenous AGEs prepared upon incubation of protein with reducing
sugar was CML (22). We thus prepared heterogenous AGE by incubation of
human IgG with ribose, 0.025 M (a relevant concentration of
reducing sugar in diabetes) for 6 weeks at 37 °C under aerobic conditions. After retrieval of this material and extensive dialysis to
remove unreacted ribose, AGE-IgG demonstrated significant
immunoreactivity with affinity purified anti-CML IgG by ELISA (data not
shown). Upon chromatography of AGE-IgG onto Affi-Gel 10 resin to which had previously been adsorbed human RAGE, material immunoreactive with
anti-CML IgG (by ELISA) was eluted in the presence of glycine buffer.
In contrast, equal volumes of glycine buffer control lacked immunoreactivity with anti-CML-IgG (data not shown). In radioligand binding assays, eluate from this Affi-Gel 10-sRAGE resin substantially suppressed binding of radiolabeled sRAGE to immobilized CML-BSA; in
contrast, glycine buffer control was without effect (Fig.
2). These data thus provided further
evidence for the specific interaction of CML-modified adducts with
RAGE.
N
-(Carboxymethyl)Lysine Adducts
of Proteins Are Ligands for Receptor for Advanced Glycation End
Products That Activate Cell Signaling Pathways and Modulate Gene
Expression*
§¶,§,
,,,,,,,, and**
College of Physicians & Surgeons, Columbia
University, New York, New York 10032 and the Institut
für Pharmazie und Lebensmittelchemie, Abteilung
Lebensmittelchemie, Universitàt Erlangen-Nürnberg,
Schuhstrasse 19, Erlangen 91052, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(carboxymethyl)lysine (CML) adducts, are
ligands of RAGE. We demonstrate here that physiologically relevant CML
modifications of proteins engage cellular RAGE, thereby activating key
cell signaling pathways such as NF-
B and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to
accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(8-9), thereby redirecting cellular function in a manner linked to
expression of inflammatory and prothrombotic genes important in the
pathogenesis of chronic disorders as apparently diverse as diabetic
macrovascular disease and amyloidosis (10-20).
- (carboxymethyl)lysine (CML)
modifications of proteins are predominant AGEs that accumulate in
vivo (21-24). Elevated serum levels of CML were demonstrated in
patients with diabetes (24-25) and renal failure (26). Importantly,
enhanced accumulation of CML was shown in vascular tissue,
atherosclerotic lesions, and glomerular tissue retrieved from diabetic
rodents and human subjects (25, 27-30). In these settings, CML adducts
co-localized with oxidation epitopes, such as malondialdehyde and
4-hydroxynonenal. These observations are consistent with the concept
that beyond processes mediating glycoxidation of proteins (31), lipid
oxidation itself triggers generation of CML (32), thereby establishing
a likely link between enhanced glycation observed in diabetic
hyperglycemia and disturbances of lipid metabolism, common to both
types 1 and 2 diabetes (33).
B, with subsequent
modulation of gene expression. Together, these findings link CML-RAGE
interaction to the development of accelerated vascular and inflammatory
complications that typify disorders in which inflammation is an
established component.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
kDa (Amersham Pharmacia Biotech). After electrophoretic separation,
contents of the gels were transferred to nitrocellulose membranes
(Bio-Rad). Unoccupied sites on the membranes were blocked in the
presence of nonfat dry milk in TBS (13.5%) for 4 h at room
temperature. Immunoblotting was performed using affinity purified
anti-CML IgG as above (14.2 µg/ml) in milk buffer (5%) for 1.5 h at 37 °C. Membranes were washed extensively in TBS containing
Tween 20 (0.1%) and membranes incubated with goat anti-rabbit IgG
labeled with horseradish peroxidase (Sigma) for 1 h at 37 °C.
Membranes were washed extensively in the above buffer, and
visualization of antibody binding was performed employing the ECL
detection system (Amersham Pharmacia Biotech). Quantitative evaluation
of band intensity was performed using Molecular Dynamics/ImageQuant (Foster City, CA).
4,000 to 5,000 cpm/ng protein. In
all cases, after radioiodination, precipitation of the radiolabeled
material in trichloroacetic acid exceeded 90%. CML modifications of
proteins as indicated or native, unmodified proteins were loaded onto
the wells of plastic dishes (Maxisorp, Nunc) (5 ng/well) in
bicarbonate/carbonate buffer, pH 9.6, and incubated for 16 h at
4 °C. Material in the wells was aspirated, and unoccupied sites were
blocked by incubation with PBS containing BSA (1%) for 2 h at
37 °C. Wells were washed twice with PBS containing octyl
-glucoside (0.005%) (Roche Molecular Biochemicals). A radioligand
binding assay was performed in PBS containing BSA (0.2%) with the
indicated concentration of 125I-human sRAGE alone or in the
presence of a 50-fold molar excess of unlabeled human sRAGE or the
indicated competitor for 2 h at 37 °C. At the end of that time,
wells were washed rapidly five times with washing buffer as above;
elution of bound material was performed in a solution containing
heparin, 1 mg/ml. Solution was aspirated from the wells and counted in
a gamma counter (Amersham Pharmacia Biotech). Equilibrium binding data
were analyzed according to the equation of Klotz and Hunston (45):
B = nKA/1 + KA, where B indicates specifically bound ligand (total binding, wells
incubated with tracer alone, minus nonspecific binding, wells incubated with tracer in the presence of excess unlabeled material), n
indicates sites/cell, K indicates the dissociation constant,
and A indicates free ligand concentration) using nonlinear
least squares analysis (Prism; San Diego, CA). Specific binding of
CML-BSA to radiolabeled RAGE was further determined by subtraction of
nonspecific binding (counts obtained upon binding of radiolabeled sRAGE
to immobilized BSA) from that obtained upon binding of radiolabeled
sRAGE to immobilized CML-BSA. In the case binding to immobilized BSA,
counts were negligible and less than 10% that observed in the presence of CML-BSA. Where indicated, pretreatment with either antibodies, soluble RAGE, or the indicated potential competitor was performed for
2 h prior to binding assay. In certain experiments, material eluted from the RAGE-Affi-Gel 10 columns (after passage of AGE-IgG) was
tested as unlabeled competitor in the binding assay; controls were
performed with equal volumes of glycine buffer. In other experiments,
isolated RAGE domains were employed as unlabeled competitors. Human
RAGE cDNA encoding the V, C1, or C2 domain was inserted into the
EcoRI site of pGEX4T vector containing GST. Fusion proteins,
V-GST, C1-GST, and C2-GST, were expressed in Escehrichia
coli, purified on a glutathione-Sepharose column, and cleaved with
thrombin (Amersham Pharmacia Biotech). RAGE domains were then purified
to homogeneity using glutathione-Sepharose and characterized by
SDS-PAGE and amino-terminal sequencing (1-2) prior to testing in the
radioligand binding assay.
B (49) as described (8-9).
Supershift assays were performed by preincubating nonimmune anti-p50,
anti-p65, or both IgG (Santa Cruz Biotechnology) with nuclear extract
for 45 min at room temperature prior to addition of radiolabeled
oligonucleotide probe. Relative intensity of the bands was determined
by densitometric analysis as above.
-glucoside (2%) for 4 h at 4 °C. The suspension was
subjected to centrifugation for 10 min at 14,000 rpm and supernatant
assessed for protein concentration (Bio-Rad). Immunoblotting was
performed after electrophoresis of 50 µg of protein/lane of SDS-PAGE
gels and transfer of gel components to nitrocellulose. Anti-VCAM-1 IgG
(0.4 µg/ml) was employed for immunoblotting as above. In other cases,
total RNA was isolated using TRIZOL (Life Technologies, Inc.) and the
concentration of total RNA determined by absorption at
A260/280. Total RNA (30 µg/lane) was subjected to electrophoresis. After electrophoretic separation on a
formaldehyde-agarose gel (0.8%), RNA was transferred onto a GeneScreen
hybridization filter (NEN Life Science Products) and linked by a UV
cross-linker (Stratagene, La Jolla, CA). The filter was then
prehybridized in QuikHyb hybridization buffer (Stratagene) for 30 min,
followed by hybridization with rat VCAM-1 cDNA probe (generously
provided by Dr. Tucker Collins) or glyceraldehyde-3-phosphate
dehydrogenase (GADPH) cDNA probe labeled with
[
-32P]dCTP using the random primer labeling system
(Stratagene) in the above hybridization buffer for 1 h. After
washing twice at room temperature (15 min/wash) with SSC (2 times)
containing SDS (0.1%) and then with SSC (0.1 time) containing SDS
(0.1%) at 60 °C for 30 min, the filter was subjected to
autoradiography at 
80 °C. cDNA for glyceraldehyde-3-phosphate
dehydrogenase was used in control studies to normalize counts in the
VCAM-1 message by densitometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2.0- and 3.3-fold increased CML immunoreactivity
of samples from diabetic and renal failure patients, respectively,
compared with normal controls (Fig.
1A). These findings were
consistent with earlier work indicating enhanced CML accumulation in
the serum of subjects with diabetes or renal failure and suggested the
relevance of albumin for modification by CML in our studies
(24-26).
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Fig. 1.
CML-modified adducts are present in human
serum albumin and bind RAGE. A, CML modification in
human serum albumin. Human serum albumin was isolated from the plasma
of age-matched healthy subjects (lane 1), diabetes
(lane 2), or renal failure (lane 3) and subjected
to immunoblotting with affinity purified anti-CML IgG as described.
Molecular weight markers were run simultaneously as indicated. Results
of densitometric analysis are shown; densitometry units in
immunoblotted albumin from normal subjects was arbitrarily defined as
"1". A representative immunoblot is shown here; this experiment was
performed on samples from 2 subjects per group with analogous results.
B, CML-modified adducts bind RAGE. CML-BSA, 310 mmol of
CML/mol of Lys, was immobilized onto the wells of plastic dishes (5 ng/well) and a radioligand binding assay performed with the indicated
amount of 125I-sRAGE alone or in the presence of a 50-fold
molar excess of unlabeled sRAGE as described above. Data were analyzed
by nonlinear least squares analysis and fit to a one-site model
according to the equation of Klotz and Hunston: Kd
76.2 ± 35 nM and capacity 2.9 ± 0.6 fmol/well. C and D, effect of anti-RAGE IgG,
sRAGE (C) or RAGE-specific domains (D) on the
binding of 125I-sRAGE to immobilized CML-BSA. Radioligand
binding assays were performed as above except that where indicated
wells were preincubated with anti-RAGE IgG, anti-CML IgG, or preimmune
IgG (70 µg/ml), or radiolabeled sRAGE was preincubated with a 10-fold
molar excess of sRAGE or free CML prior to binding assay employing
125I-sRAGE, 100 nM. D, binding
assays were performed in the presence of a 50-fold molar excess of
RAGE-specific domain, V, C1, or C2 as described. C and
D, percent maximal specific binding ± S.E. of
triplicate determinations is reported. These experiments were performed
at least three times.
76.2 ± 35 nM
(Fig. 1B). The affinity of CML-modified BSA for RAGE was
quite similar to that obtained previously employing heterogeneous AGEs, 61 ± 23 nM (1). That the observed binding was
dependent on RAGE was illustrated by inhibition of
125I-sRAGE interaction with CML-BSA in the presence of
excess sRAGE, anti-CML IgG, or anti-RAGE IgG (Fig. 1C). In
contrast, preincubation with preimmune rabbit IgG was without effect
(Fig. 1C). A polypeptide backbone for CML modification was
required for recognition by RAGE, as free CML did not effectively
compete for binding of radiolabeled sRAGE to immobilized CML-BSA (Fig.
1C).
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Fig. 2.
CML-immunoreactive structures within AGE-IgG
bind RAGE. AGE-IgG was chromatographed onto sRAGE Affi-Gel 10 resin; after washing, elution with glycine buffer revealed material
immunoreactive with anti-CML-IgG by ELISA. Eluate and glycine buffer
control were then tested for their ability to inhibit the binding of
radiolabeled sRAGE (100 nM) to immobilized CML-BSA. Percent
maximal specific binding ± S.E. of triplicate determinations is
reported. This experiment was performed twice with analogous
results.
Taken together, these studies indicated that chemically synthesized CML-modified protein, or CML adducts that form upon incubation of protein with relevant concentrations of reducing sugar under aerobic conditions, bind RAGE in a specific manner. We next sought to extrapolate these findings to those previously observed in in vitro cell culture systems to determine if CML-modified adducts ligated RAGE and modulated cellular properties.
CML-modified Adducts Mediate Cellular Activation, in Vitro Analyses
Endothelial Cells-- We previously demonstrated that heterogeneous AGEs, either those prepared in vitro or those isolated from human diabetic subjects, modulated endothelial function via engagement of RAGE (14). Since in vitro, CML-modified adducts bound RAGE, we sought to determine if these modifications were important components of heterogeneous AGEs in effecting cellular activation.
A central means by which endothelial function is modulated in the
presence of experimental diabetes is by early enhanced expression of
vascular cell adhesion molecule-1 (VCAM-1), a cell adhesion molecule
that mediates binding of mononuclear cells bearing VLA-4 to the vessel
wall (50). Incubation of HUVEC with CML-modified ovalbumin (CML-OVA),
310 mmol of CML/mol of Lys, resulted in significantly increased cell
surface expression of VCAM-1 compared with incubation with native OVA
(Fig. 3A). The central role of
RAGE in mediating interaction of CML adducts with endothelium was
demonstrated by significant suppression of CML-mediated VCAM-1
expression in the presence of either anti-RAGE IgG or excess sRAGE
(Fig. 3A). The functional significance of VCAM-1 expression
by CML-treated endothelium was shown by enhanced binding of Molt-4
cells, which bear counterligand for VCAM-1, VLA-4 (Fig. 3B).
Adherence of Molt-4 cells to stimulated endothelium was dependent on
the concentration of CML-OVA (Fig. 3B) and required
interaction of CML-modified adduct with the receptor, as shown by
dose-dependent inhibition of Molt-4 binding in the presence
of anti-RAGE F(ab')2 (Fig. 3B). Similar
inhibitory effects were observed in the presence of excess sRAGE (data
not shown). In contrast, pretreatment of CML-stimulated endothelium
with preimmune F(ab')2 had no effect (Fig.
3B).
|
Expression of VCAM-1 is subject, at least in part, to regulation at the
transcriptional level by nuclear factor-B (49). We previously
demonstrated that ligation of RAGE by heterogeneous AGEs enhanced
nuclear translocation of NF-B, as demonstrated by electrophoretic
mobility shift assay (EMSA) (8, 9). Compared with HUVEC incubated with
native OVA, nuclear extracts from cells exposed to CML-OVA, 310 mmol of
CML/mol of Lys, demonstrated a 4-fold increase in activation of
NF-B as measured by EMSA employing 32P-labeled NF-B
probe (Fig. 3C, lanes 2 and 1, respectively). That these effects of CML-OVA were largely mediated by interaction with
RAGE was confirmed by the dose-dependent inhibitory effect of anti-RAGE IgG added to the endothelium prior to CML-OVA (Fig. 3C, lanes 6 and 5). Similar inhibitory effects
were observed in the presence of excess sRAGE (data not shown). In
contrast, treatment with nonimmune IgG was without effect on
CML-OVA-mediated activation of NF-B (Fig. 3C, lane 4).
CML-mediated induction of NF-B nuclear translocation required intact
RAGE intracellular signaling, as shown by experiments using a truncated
form of the receptor, termed tail-deletion RAGE, from which the
cytosolic tail was deleted. Endothelium transfected with tail deletion
RAGE display marked suppression of NF-B activation (Fig. 3C,
lane 8) upon engagement of CML-OVA compared with cells transfected
with vector alone (Fig. 3C, lane 7), a dominant negative
(DN) effect.
Since previous studies demonstrated that the extent of CML modification
in vivo is varied (51), it was important to show that a
range of extents of protein modification by CML was capable of
mediating endothelial activation. We thus prepared and characterized CML-OVA containing 33 mmol of CML/mol of Lys, and we tested its ability
to activate NF-B. Incubation of HUVEC with this material resulted in
2.5-fold increase in nuclear translocation of NF-B by EMSA,
compared with incubation in the presence of native OVA (Fig.
3D, lanes 1 and 2, respectively). That
this was dependent on ligation of RAGE was demonstrated by suppressed
activation of NF-B by CML-OVA in the presence of anti-RAGE IgG but
not by nonimmune IgG (Fig. 3D, lanes 3 and
4, respectively). Similar inhibitory effects were noted with
excess sRAGE (data not shown).
Taken together, these data suggested that CML-modified proteins were specific AGEs that mediate cellular activation in endothelium, at least in part, via RAGE.
Vascular Smooth Muscle Cells--
We next sought to test the
hypothesis that CML-modified proteins interacted with RAGE on vascular
smooth muscle cells (VSMC), as such cells have been implicated in
vascular perturbation in diabetes and renal failure. In VSMC,
activation of NF-B is a potent means by which regulation of
cytokines and vasoregulatory mediators is affected (52-53).
We first tested varied extents of CML modification in order to
determine if their interaction with VSMC RAGE resulted in activation of
NF-B. Incubation of VSMC with CML-BSA (90, 160, or 310 mmol CML/mol
Lys) resulted in 3.2-, 2.6-, and 3.2-fold induction of nuclear
translocation of NF-B (Fig. 4A,
lanes 3-5, respectively) compared with incubation in the presence
of native BSA (Fig. 4A, lane 2). In VSMC treated with
CML-BSA, 90 mmol of CML/mol of Lys, activation of NF-B was dependent
on interaction with RAGE, as demonstrated by suppression in the
presence of either anti-RAGE IgG or excess sRAGE (Fig. 4B, lanes
5 and 6, respectively). In contrast, nonimmune IgG was
without effect (Fig. 4B, lane 4). Furthermore, incubation of
VSMC with CML-OVA, 33 mmol of CML/mol of Lys, resulted in 3.1-fold
increase in activation of NF-B compared with cells incubated with
native OVA (Fig. 4C, lanes 3 and 2,
respectively). That these effects were mediated by RAGE was indicated
by marked suppression of CML-OVA-mediated activation of NF-B in the
presence of anti-RAGE IgG (Fig. 4C, lane 5). Similar suppressive effects were noted in the presence of excess sRAGE (data
not shown). In contrast, incubation with nonimmune IgG was without
effect (Fig. 4C, lane 4).
|
These data indicated that at low levels of CML modification,
interaction of these modified adducts with RAGE on VSMC resulted in
activation of NF-B, thereby providing a potent mechanism by which
VSMC function may be altered in the presence of CML adducts of proteins.
Mononuclear Phagocytes--
Previous studies from our laboratory
indicated that MP properties are significantly modulated upon ligation
of RAGE by heterogeneous AGEs, either those prepared in
vitro or those derived from in vivo sources, such as
AGEs isolated from diabetic plasma, or
AGE-2-microglobulin isolated and purified from the
urine/plasma of subjects with DRA (16). It was thus important to
determine if CML adducts, present in these preparations, could activate
RAGE on MPs causing cellular migration and activation in this setting
(54). Furthermore, since recent studies have indicated that CML
modifications may result not only from glycation/oxidation of proteins,
but also secondary to modification of lipids, a possible role CML-RAGE interaction in lipid-rich foam cells and atherosclerotic plaques, even
in euglycemia, might occur (5).
We thus tested the ability of CML-modified adducts to activate
monocytes. In modified chemotaxis chambers, compared with native OVA,
addition of CML-OVA, 310 mmol of CML/mol of Lys, resulted in enhanced
migration of Molt-4 cells, mononuclear type cells that bear cell
surface RAGE (Fig. 5A, lines 1 and 2, respectively). The effects of CML-OVA were mediated
by cellular RAGE as preincubation of Molt-4 cells with anti-RAGE
F(ab')2 resulted in suppression of migration in a
dose-dependent manner (Fig. 5A, lines 4 and 5); preincubation with nonimmune F(ab')2 was
without effect (Fig. 5A, line 3). Similarly, preincubation
of CML-OVA with excess sRAGE resulted in dose-dependent
suppression of Molt-4 migration (Fig. 5A, lines 6 and
7). Intact RAGE signaling was required for these CML-mediated effects on migration as transient transfection of Molt-4
cells with a construct expressing tail deletion RAGE resulted in
significant suppression of Molt-4 migration in response to CML-OVA;
mock transfection was without effect (Fig. 5A, lines 9 and
8, respectively).
|
It was important to test lower extents of modification of CML-OVA in order to determine their ability to modulate Molt-4 migration. These studies revealed that addition of CML-OVA, 33 mmol/mol Lys, to chemotaxis chambers resulted in increased migration of Molt-4 cells compared with native OVA (Fig. 5B, lines 2 and 1, respectively). These effects were mediated by ligation of RAGE as demonstrated by suppression of migration in the presence of either anti-RAGE F(ab')2 or excess sRAGE (Fig. 5B, lines 4 and 5, respectively). In contrast, incubation with nonimmune F(ab')2 was without effect (Fig. 5B, line 3).
Recent reports have indicated that in lens protein and skin collagen retrieved from elderly human subjects, extent of modification by CML adducts is in the approximate range of 4.95 and 1.70 mmol of CML/mol of Lys, respectively (51). The extent of CML modification is likely to be higher in atherosclerotic plaques and inflamed joints in DRA, for example, due to the higher concentration of highly bioactive species involved in CML formation compared with slow protein turnover observed in lens or skin collagen protein. We tested the ability of CML-OVA, 0.8 mmol of CML/mol of Lys, to engage RAGE on mononuclear cells and to redirect cellular function. Compared with native ovalbumin, addition of these minimally modified CML-OVA adducts to chemotaxis chambers caused a significant increase in cell migration (Fig. 5C, lines 1 and 2, respectively). These effects were mediated by RAGE, as indicated by suppression of CML-OVA-mediated migration in the presence of either anti-RAGE F(ab')2 or excess sRAGE (Fig. 5C, lines 4 and 5, respectively).
Consistent with these findings, exposure of MP-like BV2 macrophages to
CML-OVA resulted in 3.5-fold increase in nuclear translocation of
NF-B by EMSA, compared with incubation in the presence of native OVA
(Fig. 5D, lanes 1 and 2, respectively).
Activation of NF-B by CML adducts was due to ligation of RAGE as
exemplified by significant suppression of NF-B in the presence of
anti-RAGE IgG, 70 µg/ml (Fig. 5D, lane 6), but not in the
presence of either lower concentrations of anti-RAGE IgG, 0.7 µg/ml,
or nonimmune IgG (Fig. 5D, lanes 5 and 4,
respectively). Supershift assays with anti-p50 and anti-p65 IgG
demonstrated that the NF-B complex activated upon ligation of BV-2
RAGE by CML-OVA was composed of both p50 and p65 (Fig. 5D,
lanes 8-10). Similar inhibitory effects were observed in
the presence of transient transfection of construct encoding DN-RAGE
(Fig. 5E, lane 2) or excess sRAGE (data not shown) but not
by nonimmune IgG (Fig. 5E, line 3).
CML-modified Adducts Mediate Cellular Activation, in Vivo Analyses
The results of in vitro analyses suggested that CML
adducts, at physiologically relevant levels of modification, were
capable of mediating cellular activation in a range of cell types. A
central test of this hypothesis was the ability of CML-modified adducts to modulate cellular properties in vivo. As a first test of
these concepts, we infused CML-BSA, 310 mmol of CML/mol of Lys, into immunocompetent, non-diabetic mice. Initial studies indicated that in
comparison to infusion of native BSA, CML-BSA induced a 46-fold
increase in mRNA for VCAM-1 in lung tissue (Fig.
6A, lanes 2 and 1,
respectively). To determine if this was associated with modulation of
VCAM-1 protein levels in the lung, immunoblotting was performed.
Indeed, infusion of CML-BSA into mice resulted in a 3.6-fold
increase in expression of VCAM-1 protein compared with infusion of
native BSA (Fig. 6B, lanes 2 and 3,
respectively). That this was dependent on ligation of vascular RAGE in
the lung by CML-BSA was demonstrated by experiments in which mice were pre-infused with either anti-RAGE IgG or excess sRAGE; in both cases,
significant suppression of CML-mediated expression of VCAM-1 in the
lung was noted (Fig. 6B, lanes 4 and 6,
respectively). In contrast, infusion of nonimmune IgG had no effect on
CML-mediated modulation of VCAM-1 expression (Fig. 6B, lane
5). These findings illustrate a mechanism by which engagement of
vascular RAGE by CML-modified adducts enhances expression of VCAM-1
and, likely, increases adherence of proinflammatory mononuclear cells
to the vessel wall.
|
| |
DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Increased recognition of specific AGEs and the multiple means by
which they might form in diverse settings such as hyperglycemia, renal
failure, and inflammation has highlighted the importance of determining
mechanisms by which each might modulate cellular properties, either by
receptor-dependent or -independent pathways. In this
context, previous studies indicated that heterogenous AGEs, especially
those derived from in vivo sources, were signal-transducing ligands of RAGE. Specifically, interaction of
AGE-2-microglobulin, isolated from the urine of subjects
with renal failure and DRA, with cellular RAGE resulted in enhanced MP
migration and generation of proinflammatory cytokines such as tumor
necrosis factor- (16). Since recent studies have shown
AGE-2M itself to be quite heterogeneous, composed at
least in part of AGEs such as pentosidine, CML, and imidizalone
(55-57), it was important to begin to delineate those specific AGEs
that interacted with RAGE.
In the present work, we have identified that CML adducts, dominant AGEs within the tissues, are signal-transducing ligands for RAGE in vitro, in endothelial cells, mononuclear phagocytes, and vascular smooth muscle, and in vivo, upon infusion into mice. Importantly, we tested varied extents of CML modification to demonstrate that the CML adducts thus formed exerted specific and receptor-dependent effects. In this context, physiologically relevant levels of CML modification of ovalbumin were capable of modulating MP function. Such levels of modification parallel those reported in relatively inert tissue derived from elderly human subjects, such as skin collagen and lens protein (51). We speculate that in a highly biologically active milieu, such as lipid-laden atherosclerotic fatty streaks and plaques and chronically inflamed joints, levels of CML modification will likely exceed those tested here.
In proper context, however, levels of CML-modified adducts are low, even in tissue from subjects with diabetes or renal failure (51). We hypothesize that the inexorable accumulation of AGEs, via their interaction with RAGE, causes a change in cellular homeostasis priming mechanisms capable of triggering full-blown cellular activation. Such chronic AGE engagement of RAGE causes a shift in the basal state, favoring cellular activation with subsequent environmental challenge. Thus, AGE-RAGE interaction may be considered the first hit in a "two-hit" model of diabetic complications. In wound repair, the second hit comprises the presence of foreign bodies and bacterial pathogens. In atherosclerotic vasculature, the second hit is an environment enriched in modified lipoproteins and macrophages. Juxtaposition of these two mechanisms results in an exaggerated cellular response, ultimately compromising reparative processes in diabetic tissues. Thus, as opposed to septic shock, in which highly potent lipopolysaccharide released by Gram-negative bacteria engages cellular sites, resulting in striking rapid activation and destabilization of homeostasis, in settings such as diabetes, renal failure, and chronic arthritis, long term, yet unrelenting, chronic cellular activation ensues, eventuating in irreversible tissue injury.
An important means by which AGEs modulate cellular properties is via
generation of enhanced oxidant stress, manifested by increased
transcripts for heme oxygenase and nuclear translocation of NF-B (8,
9), the latter a central cell signaling molecule whose activation is
linked to transcriptional regulation of a range of proinflammatory
genes, including RAGE itself (58).
We speculate that these events, significantly suppressed in the presence of antioxidants (8, 9) and mediated at least in part via RAGE, are early and highly influential steps in processes which, long term, contribute to cellular dysfunction. Consistent with these concepts, in experimental diabetes, AGEs begin to form within weeks of hyperglycemia (Refs. 19, 20, and 59). Thus, although evidence does not exist for globally enhanced oxidant stress in diabetes, as measured by lack of increased ortho-tyrosine and methionine sulfoxide in diabetic collagen (60), we speculate that in tissues such as the vessel wall and periodontium, sustained exposure to damaging substrates typified by high levels of modified lipid and Gram-negative bacteria, respectively, may yield CML adducts and thus mediate chronic local oxidant stress via RAGE. These local changes critically modulate cellular properties. Consistent with this view, therefore, lack of increased CML adducts in diabetic urine (61) may be insensitive in detecting locally relevant enhanced accumulation and oxidant stress. Indeed, support for the concept that chronic generation of CML-modified adducts at locally relevant sites in diabetic organisms contributes, at least in part, to mechanisms underlying complications, may be deduced from the findings that the extent of diabetic complications, such as those in the vasculature and retina, correlate with the degree of CML accumulation (62-63).
The findings presented here do not rule out other specific AGE products of glycation or oxidation, such as pentosidine, pyralline, methylglyoxal, and imidizalone (64-66) as signal-transducing ligands of RAGE. Indeed, although certainly present in the tissues at even lower degrees than CML, it is possible that locally relevant concentrations of these modified adducts may engage cellular RAGE to redirect cellular properties. Similarly, our findings do not rule out the presence of other receptors or cellular interaction sites for CML adducts. Although our studies suggest that RAGE is a receptor for CML, it is possible that other receptors for AGE (67-69) may also engage CML-modified adducts. Indeed, it will be of particular interest to determine if the macrophage scavenger receptor plays a role in removal/detoxification of CML adducts, as recent views have emerged suggesting that overtaxed detoxification pathways lead to accelerated accumulation of toxic AGEs and highly reactive carbonyl compounds. Ultimately, it is speculated that these products exacerbate tissue injury, especially in the later stages of diabetes or renal failure (70-71). Alternatively, different cellular receptors for AGEs may recognize diverse AGEs. For example, the macrophage scavenger receptor has been reported to bind glycoaldehyde (72), which does not bind RAGE.
Certainly, in complex disorders such as renal failure and diabetes,
numerous factors likely converge in the development of cellular
abnormalities. In diabetes, hyperglycemia directly results in
activation of protein kinase C, especially the II isoform, leading
to cellular dysfunction (73-74). In addition, accelerated generation
and accumulation of products of the sorbitol/polyol pathway are
implicated in the pathogenesis of diabetic complications, especially
those of the retina and peripheral nerve (75). Recent observations
suggesting that polyol metabolites may lead to generation of AGEs (76)
suggest that, not unexpectedly, seemingly diverse pathways may converge
and thus collaboratively contribute to cellular perturbation. These
findings indeed parallel the diverse means by which CML adducts may
form: from glycation/oxidation of proteins to lipid oxidation and
myeloperoxidase-driven modification of amino acids and related structures.
Taken together, our observation that physiologically relevant extents
of CML modification in proteins are signal-transducing ligands for RAGE
transforms these adducts from inert biomarkers of aging and chronic
disease to bioactive species capable of altering properties of cells
critically perturbed in inflammatory milieu. Our findings provide a
contributory mechanism for the chronic, yet unrelenting progressive
vascular and inflammatory cell dysfunction that typifies the
complications of diverse disorders, such as diabetes, renal failure,
chronic inflammation and Alzheimer's disease, in which CML adducts
accumulate and the expression of RAGE is enhanced. Thus, identification
of specific molecular structures to inhibit interaction of CML-modified
adducts with RAGE may provide a potent means to suppress cellular
activation and, thereby, limit long term tissue injury in chronic diseases.
| |
FOOTNOTES |
|---|
* This work was supported in part by the Surgical Research Fund of the College of Physicians and Surgeons, Columbia University, United States Public Health Service Grants DK52495, HL56881, HL60901, AG00602, DE11561, and AG00690, the Juvenile Diabetes Foundation International, the American Heart Association, New York affiliate, and the Council for Tobacco Research.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.
§ Both authors contributed equally to this work.
¶ Recipient of a grant from the Deutsche Forschungsgemeinschaft.
** To whom correspondence should be addressed: College of Physicians and Surgeons, Columbia University, 630 West 168th St., P&S 17-501, New York, NY 10032. Tel.: 212-305-6406; Fax: 212-305-5337; E-mail: ams11@columbia.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
AGEs, advanced
glycation end products;
AGE-2M, AGE-2-microglobulin;
BSA, bovine serum albumin;
CML, N- (carboxymethyl)lysine;
DN, dominant
negative;
DRA, dialysis-related amyloidosis;
endothelial cell, ELISA,
enzyme-linked immunosorbent assay;
EMSA, electrophoretic mobility shift
assay;
GST, glutathione S-transferase;
HUVEC, human
umbilical vein endothelial cells;
KLH, keyhole limpet hemocyanin;
MP, mononuclear phagocyte;
OVA, ovalbumin;
PBS, phosphate-buffered saline;
sRAGE, soluble RAGE;
RAGE, receptor for AGE;
TBS, Tris-buffered
saline;
VSMC, vascular smooth muscle cells;
PAGE, polyacrylamide gel
electrophoresis.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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C, EMSA. Rat VSMC were treated with the indicated
mediators for 6 h. A, VSMC were treated with CML-BSA,
90, 160, or 310 mmol of CML/mol of Lys (lanes 3-5).
B, VSMC were treated with CML-BSA, 90 mmol of CML/mol of Lys
in the presence of anti-RAGE IgG, nonimmune IgG, or excess sRAGE as
above. C, VSMC were treated with CML-OVA, 33 mmol of CML/mol
of Lys, in the presence of anti-RAGE IgG or nonimmune IgG. In all
cases, after incubation as indicated, nuclear extracts were prepared
and EMSA performed employing radiolabeled probes for NF-
