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Laboratoire de Recherche en Biologie Vasculaire et Cellulaire, EA 1557, Université Paris 7, Hôpital Lariboisière, Paris, France (C.R., O.C., M.-P.W., J.-L.W.), Institut National de la Santé et de la Recherche Médicale Unité 26, Hôpital Fernand Widal, Paris, France (C.R., J.-M.S.), Berlex Biosciences, Richmond, California 94804 (M.N., E.L., J.M., L.Z.), and Department of Physiology, Medicine, and Surgery, Columbia University, College of Physicians and Surgeons, New York 10032 (A.-M.S.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Vascular dysfunction in patients with diabetes mellitus is related to
advanced glycation end product (AGE) formation. We previously showed
that AGEs produce an increase in vascular permeability and generated an
oxidant stress after binding to the receptor (RAGE) present on
endothelium. RAGE, a 35-kDa protein that belongs to the immunoglobulin
superfamily, has been cloned from a rat lung cDNA library, and
recombinant rat soluble RAGE (rR-RAGE) has been produced in insect
cells. The sequence of RAGE is highly conserved between human and rat.
We studied the biological effect of rR-RAGE and pharmacokinetics of
125I-rR-RAGE after intravenous or intraperitoneal
administration in normal and streptozotocin-induced diabetic rats.
rR-RAGE prevented albumin or inulin transfer through a bovine aortic
endothelial cell monolayer, restored the hyperpermeability observed in
diabetic rats or induced in normal rats by diabetic rat red blood
cells, and corrected the reactive oxygen intermediate production after intravenous or intraperitoneal administration. After intravenous injection of 125I-rR-RAGE, the distribution half-life was
longer (p 
0.01) in diabetic (0.15 and 4.01 hr) than in normal (0.02 and 0.21 hr) rats, as was the case for the
elimination half-lives (diabetic, 57.17 hr; normal, 26.02 hr;
p 0.01). Distribution volume was higher in
diabetic than in normal rats (6.94 and 3.24 liter/kg, respectively;
p = 0.049). Our study showed that rR-RAGE was
biologically active in vivo and slowly cleared, which
suggests it could be considered as a potential therapy.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Vascular dysfunction is one of the main complications in patients with diabetes mellitus. Several hypotheses have been proposed to explain vascular abnormalities linked to hyperglycemia, including sorbitol toxicity (1), diacylglycerol protein kinase activation (1), AGE formation (1, 2), and reactive oxygen species production (3). AGEs are the ultimate products of a nonenzymatic glycation of primary amino groups on proteins or lipids. These compounds are yellow-brown with characteristic fluorescence spectrum (1-3). The RAGE has been purified and cloned; it is a protein of 35 kDa that belongs to the immunoglobulin superfamily. It is composed of extracellular domains (one V-type domain followed by two C-type domains), a single transmembrane spanning domain, and a highly charged cytosolic tail (4). The RAGE is present on several cell types, including ECs, smooth muscle cells, monocytes/macrophages, cardiac myocytes, neural tissue, and hepatocytes (5).
We recently showed that AGE engagement to a receptor present on ECs produces an oxidant stress and results in an increased vascular permeability (6, 7). We showed that sRAGE form could be used to prevent the consequences of the presence of AGE in the plasma and demonstrated that the newly described sRAGE prevents the effects of AGEs on EC functions and on the hyperpermeability observed in streptozotocin-induced diabetic rats or in normal rats that had been transfused with diabetic rat RBCs (7). sRAGE acts by binding to AGEs, which blocks their interaction with endogenous RAGE and restores vascular functions (7).
Because we previously established that AGE-RBCs induced an oxidant stress and were responsible for the increase in vascular permeability, in the current study we explored the properties of soluble rR-RAGE produced in insect cells on vascular permeability and reactive oxygen intermediate formation. We observed that as was the case with purified RAGE, rR-RAGE prevented the increased albumin or inulin transfer through a bovine aortic EC monolayer and corrected the hyperpermeability found in diabetic rats or induced by infusion of diabetic rat RBCs into normal rats. Because rR-RAGE seems to be efficient, we studied the pharmacokinetic parameters to evaluate the possible therapeutic use of such a recombinant protein.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Cloning, Expression, and Purification of Soluble rR-RAGE
Full-length rat RAGE cDNA was cloned by screening a lung cDNA
library (Clontech Laboratories, Palo Alto, CA) with a 187-bp DNA probe
corresponding to the signal sequence of human RAGE (8). Filters were
hybridized in a buffer of 50% formamide/5× SSC/10 mM
sodium phosphate, pH 6.4, and 0.1% SDS, 1× Denhardt's solution, and
50 µg/ml sonicated sperm DNA for 16 hr at 42°. Filters were washed
in 6× SSC/0.1% SDS four times for 15 min each at room temperature, followed by washing in 1× SSC/0.1% SDS at 48° and exposure for 48 hr to X-ray film. Approximately 1 × 106 plaques were
screened. The longest insert DNA from positive plaques was sequenced
according to the dideoxy chain termination method (9). A DNA fragment
coding for soluble RAGE was obtained using a polymerase chain reaction
with primers 5
-CATGCCAGCGGGGACAGCAGCT-3 and
5-TAGCGTACCCAGCCCAGACTC-3. The polymerase chain reaction product was
first subcloned into the pCR II vector (InVitrogen, San Diego, CA), and
the EcoRI fragment of the resultant plasmid was cloned into
the pBacPAK8-based vector (Clontech), which contained an in-frame stop
codon.
Baculovirus expression of rR-RAGE was performed by cotransfecting the
plasmid pBacPAK8/RAGE with a linearized BacPAK6 viral DNA (Clontech)
into Sf9 cells according to the manufacturer's instructions. Recombinant plaques were identified and purified by their
-galactosidase-negative phenotype. rR-RAGE was purified from
Sf9 media by chromatography on a SP Sepharose fast-flow
column (Pharmacia, Uppsala, Sweden) equilibrated with sodium phosphate (20 mM, pH 7.5) and eluted with a salt gradient (0-0.5
M NaCl in the equilibration buffer). Fractions containing
rR-RAGE were applied to Superdex 200PG (Pharmacia) in PBS to obtain an
homogeneous sample of ~35 kDa on SDS-PAGE. This protein corresponded
to that expected for the extracellular domain (2).
Radiolabeling of Proteins
rR-RAGE, albumin (Sigma-Aldrich Chimie, Saint Quentin Fallavier,
France), and murine Fab were labeled with 125I-Na according
to the iodogen method (10). One hundred micrograms of protein was
incubated with 0.5 mCi of 125I-Na in Eppendorf tubes coated
with 10 µg of iodogen reagent
(1,3,4,6-tetrachloro-3
,6-diphenylglycouril; Sigma-Aldrich Chimie)
for 5 min at room temperature. Free iodine was removed by
chromatography on a Sephadex G 25-M PD 10 column (Pharmacia) equilibrated with PBS. Precipitation of the iodinated protein by TCA (10%) at +4° gave >95% of bound iodine. Specific activities were in the range of 0.9 µCi/µg for rR-RAGE, 2.5 µCi/µg for albumin, and 1.15 µCi/µg for murine Fab. The
purities of 125I-rR-RAGE, 125I-albumin, and
125I-Fab preparations were also analyzed by SDS-PAGE.
Human Diabetic RBCs, Cultured ECs, and In Vitro Permeability Assays
RBCs were obtained from normal subjects and diabetic patients in
accordance with the provisions of the Declaration of Helsinki and the
rules of our institution. Diabetic patients and normal subjects were
selected according to criteria previously described (7). In
vitro permeability assays were performed according to our previous
protocol (7). ECs were incubated with medium alone, normal RBCs, or
diabetic RBCs (2.5 × 109 cells/ml) for 24 hr. To
study the effect of rR-RAGE, ECs were incubated with diabetic RBCs with
or without rR-RAGE (60 µg/ml). Permeability coefficient (P) was
determined by using 125I-albumin and 3H-inulin
in medium: P = J × (1/A) × [1/(CT 
CB)], where J is the flux of molecules across the filter,
A is the surface area, and C is the concentration of tracer in top
(CT) and bottom (CB) chambers (7, 11).
In Vivo Permeability Studies
In vivo permeability studies were carried out as previously described (7). Normal rats received either normal RBCs, diabetic RBCs, or diabetic RBCs plus rR-RAGE (5.15 mg/kg), and diabetic rats received the same dose of R-RAGE by an intravenous bolus. In vivo permeability in normal and diabetic rats was determined using the TBIR method (7, 12).
TBARS Assay
The effect of rR-RAGE (2.25 mg/kg) on the oxidant stress was
determined by measurement of TBARS, which form a fluorescent compound
after a reaction with thiobarbituric acid (13) (Sobioda, Grenoble,
France). Serum was stored at 20° until analysis; before assay,
t-butyl-4-hydroxyanisole (Sigma-Aldrich Chimie) was added to
the specimens in a final concentration of 10 µmol/liter to prevent
artificial auto-oxidation.
Pharmacokinetics of 125I-rR-RAGE or Murine 125I-Fab
Plasma, urine, and tissue kinetics of 125I-rR-RAGE
were performed in normal and diabetic male Wistar rats (200-250 g)
placed into metabolic cages after the injection. Animals had free
access to food and water before the experiments and were anesthetized with ether before intravenous or intraperitoneal administration of
125I-rR-RAGE (250 µg/kg, volume 1 ml). For intravenous
administration, 125I-rR-RAGE was injected in bolus via the
femoral vein, and blood samples (50 µl) were collected in heparinized
tubes via the tail vein at 2, 5, 10, 30, and 45 min and 1, 1.5, 2, 3, 6, 8, 24, 30, 48, 54, 72, and 96 hr. After intraperitoneal
administration, blood samples were collected at the same times except
that the first sample was taken at 10 min. The blood hematocrit
measured at 6, 24, 54, and 96 hr after 125I-rR-RAGE
injection did not differ from physiological values.
Rats were killed at 96 hr, and radioactivity in organs (kidney, liver, spleen, intestine, vena cava, aorta, lung, and skin) was determined. Furthermore, tissue distribution of 125I-rR-RAGE was studied in normal and diabetic rats at the time corresponding to 87.5% of the 125I-rR-RAGE distribution (i.e., 38 min and 12 hr for normal and diabetic rats, respectively). The amount of 125I-rR-RAGE determined in organs was corrected by the presence of radioactivity in the residual blood remaining in the tissue (14). Urine was collected at 1 and 2 hr and then at each blood collection. Blood samples were centrifuged at 3000 × g for 10 min, and plasma was isolated. The TCA-precipitable fraction of plasma (20 µl) and urine (150 µl) was counted in a gamma counter (Minaxi Gamma 5000; Packard Instruments, Rungis, France). Creatinine clearance in normal and diabetic rats was calculated using creatinine concentration in urine and plasma at the midpoint of the urine collection interval.
To assess whether 125I-rR-RAGE could induce the urinary excretion of AGEs in diabetic rats, urinary samples were incubated with anti-AGE antibodies (molar ratio of 125I-rR-RAGE to anti-AGE antibodies = 1) for 1 hr at +37° and then overnight at +4°. The 125I-rR-RAGE/AGE complexes were then separated from 125I-rR-RAGE unbound with polyethylene glycol precipitation (7% of polyethylene glycol in borate buffer).
Pharmacokinetics of a molecule belonging to the immunoglobulin superfamily was also performed to assess whether pharmacokinetic parameters determined in diabetic rats could be influenced by AGEs or by the hyperpermeability observed in diabetic animals. Pharmacokinetics of a monoclonal murine 125I-Fab (250 µg/kg) was performed in normal (five rats) and diabetic (three rats) rats after intravenous bolus injection according to the experimental protocol described for 125I-rR-RAGE.
Pharmacokinetic Analysis
Plasma concentration-time data for 125I-rR-RAGE were
analyzed using Siphar Software (SIMED, Créteil, France) and
fitted to a three-compartment model. We used the Powell minimization
algorithm, and the model was fitted to data using weighed least-squares
with a 1/y2 weighing factor. To select the most
appropriate model, an examination of the standard deviation of
distribution and elimination rate constants (
1, 2,
z) and half-lives (t1/21,
t1/22,
t1/2z) was performed. In addition,
the likelihood test and Akaike criterion were tested. The distribution
(t1/21 and t1/22)
and terminal (t1/2z) half-lives
were calculated as 0.693/. The area under the plasma
125I-rR-RAGE concentration-time curve from zero to infinity
was determined by linear trapezoidal estimation from 0 to the last
measured time with extrapolation to infinity by adding the value of the
last measured plasma concentration divided by the terminal rate
constant. The CL, steady state volume of distribution, Vz,
and mean residence time were calculated using standard equations (15).
The maximal concentration (Cmax) and
corresponding experimental time (Tmax) after the
intraperitoneal injection of 125I-rR-RAGE were
experimentally observed values. The absorption rate coefficient,
distribution and elimination half-lives
(t1/21, t1/2z), and absolute
bioavailability (F) were determined using Siphar Software. Plasma
concentration-time data were fitted to a two-compartment model with the
Powell minimization algorithm and a 1/y2
weighing factor. The absolute bioavailability was calculated by the
area ratio method using the mean values of pharmacokinetic parameters
(16).
The 125I-rR-RAGE CLR was calculated between 0 and 96 hr after 125I-rR-RAGE injection (15), and CLR mechanisms were analyzed by plotting the urinary excretion rate of 125I-rR-RAGE versus the corresponding plasma concentration at the midpoint of the urine collection interval. These concentrations were calculated from the plasma 125I-rR-RAGE concentration-time curve. CLR of 125I-rR-RAGE was also estimated after linear regression analysis on the basis of the slope of the urinary excretion rate versus plasma concentration.
Plasma pharmacokinetics of 125I-Fab was described by a two-compartment model, and pharmacokinetic parameters were calculated as previously calculated for 125I-rR-RAGE.
Identification of 125I-rR-RAGE
Immunoidentification of 125I-rR-RAGE. Specific anti-RAGE antibodies (6) were used in an immunoprecipitation assay to determine the immunoreactive fraction of radiolabeled rR-RAGE before and after injection in rats. 125I-rR-RAGE sample (25 µl) was incubated with the anti-RAGE antibodies (50 µl; dilution, 1/12) and PBS (225 µl) for 1 hr at 37° and 1 hr at 4°. Polyethylene glycol 8000 (7% in borate buffer) (Sigma-Aldrich Chimie) was added and incubated overnight. The precipitate isolated by centrifugation was counted in a gamma counter (Packard Instruments).
SDS-PAGE analysis. Plasma and urine samples containing 125I-rR-RAGE were analyzed by SDS-PAGE on a 15% acrylamide gel under nonreducing conditions. Radiolabeled proteins and metabolites were autoradiographed using X-ray film (Amersham, Les Ulis, France) and intensifying screens for 8 weeks. The migration zone of labeled proteins or metabolites were compared with prestained standards (phosphorylase b, 101 kDa; bovine serum albumin, 83 kDa; ovalbumin, 50.6 kDa; carbonic anhydrase, 35.5 kDa; soybean trypsin inhibitor, 29.1 kDa) (BioRad, Paris, France).
Statistical Analysis
Results are presented as mean ± standard error. Two-way analysis of variance followed by parametric Dunnett's test in the event of significant differences was used to compare permeability of ECs in the presence of normal or diabetic RBCs and the results of in vivo permeability studies. Mean values of pharmacokinetic parameters were compared using the nonparametric Mann-Whitney two-sample test.
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Cloning of Full-Length Rat RAGE cDNA
To isolate full-length rat RAGE cDNA, a rat lung cDNA library was screened with a probe corresponding to the signal sequence of human RAGE with moderately high stringency. Approximately 100 positive clones were obtained from 1 million screened plaques; this is similar to the number of positive clones observed in a human lung cDNA library (4). Several clones were further characterized. The DNA sequence and the deduced amino acid sequence of the longest insert are shown in Fig. 1. Rat RAGE has 81% sequence similarity to that of human RAGE at the nucleic acid level and 88% at the amino acid level. All the cysteine residues are conserved between rat and human RAGE, indicating their structural importance.
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Expression and Characterization of rR-RAGE
To express a large amount of soluble rR-RAGE for the permeability studies, a DNA fragment coding for sRAGE was obtained using a polymerase chain reaction and cloned into a baculovirus expression vector. After the cotransfection of plasmid pBacPAK8/RAGE with BacPAK 6 viral DNA into Sf 9 cells, recombinant plaques were identified, and media were screened for the expression of recombinant protein by Western blotting using monoclonal antibody raised against rR-RAGE.
rR-RAGE was purified from the conditioned media (Fig. 2A, lane 02) on an SP Sepharose fast-flow column and equilibrated with 20 mM sodium phosphate buffer, pH 7.5. Using a 0-0.5 M salt gradient, rR-RAGE was eluted from the column between fractions 17-28 (Fig. 2A, lanes 03-14). Contaminants were removed from the pooled fractions using gel filtration chromatography (Fig. 2B). The major band corresponds to purified rR-RAGE, with a molecular mass of 42 kDa. The faint band seen slightly below 30 kDa protein marker is a cleaved form of rR-RAGE because it also binds to monoclonal anti-RAGE antibody (result not shown). The fractions containing rR-RAGE were pooled and used to test the effect of sRAGE on permeability.
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In Vitro Permeability.
The transfer of
molecular tracers (125I-albumin and 3H-inulin
in Fig. 3, A and B, respectively) through the EC barrier
was similar in minimal essential medium plus albumin or in the presence
of normal RBCs. The addition of diabetic RBCs to ECs significantly increased by 2-fold (p 0.001) and 1.3-fold
(p 0.01) the permeability to
125I-albumin or 3H-inulin, respectively.
Preincubation of RBCs with rR-RAGE prevented the effect of diabetic
RBCs on permeability of the EC monolayer; this effect was dependent on
the rR-RAGE concentration for transfer of 3H-inulin.
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In Vivo Permeability. Diabetic rats had a vascular hyperpermeability to albumin compared with normal rats. The vascular leakage was more pronounced in skin, intestine, kidney, vena cava, and heart, being 2.8-, 2.6-, 2.4-, 2.2-, and 2.1-fold increased, respectively (Fig. 4A). A bolus injection of rR-RAGE (5.15 mg/kg) corrected the enhanced permeability observed in diabetic rats. The correction of hyperpermeability was observed 1 hr after rR-RAGE injection and was higher in skin, intestine, vena cava, heart, and aorta (Fig. 4A). Infusion of diabetic RBCs in normal rats increased the permeability to albumin compared with normal RBCs infused in normal rats (Fig. 4B). The administration of rR-RAGE with the injection of diabetic RBCs prevented the increase of albumin transfer and extravasation (Fig. 4B) in skin, heart, brain, kidney, and vena cava. The results are in agreement with our previous study (7).
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Effect of rR-RAGE on Oxidant Stress. Normal rats had significantly lower plasma TBARS levels than diabetic rats (1.76 ± 0.08 and 2.76 ± 0.12 µmol/liter, respectively; p = 0.002). Both intravenous and intraperitoneal administration of rR-RAGE significantly reduced plasma TBARS levels (2.08 ± 0.09 µmol/liter, p = 0.011, and 2.12 ± 0.04 µmol/liter, p = 0.007, respectively). This showed that rR-RAGE was efficacious in reducing oxidant stress after intravenous or intraperitoneal injection.
Plasma and Urine Pharmacokinetics. After intravenous bolus, 125I-rR-RAGE plasma disposition in normal or diabetic rats was described by a three-exponential decay (rats 25 and 26, respectively; Fig. 5). The mean pharmacokinetic parameters are presented in Table 1. Distribution and elimination half-lives were significantly different in normal and diabetic rats. A rapid initial distribution phase was observed in normal and diabetic rats (0.02 ± 0.01 and 0.15 ± 0.03 hr, respectively; p = 0.008), followed by a slower distribution phase in diabetic rats compared with that in normal rats (4.01 ± 0.87 and 0.21 ± 0.07 hr, respectively; p = 0.008). Finally, plasma concentrations decreased with different elimination half-lives of 26.02 ± 2.36 and 57.17 ± 11.62 hr for normal and diabetic rats, respectively (p = 0.008). The Vz of 125I-rR-RAGE was significantly higher in diabetic rats than in normal rats (6.9 ± 1.8 and 3.24 ± 0.76 liter/kg, respectively; p = 0.049). Total body clearance of 125I-rR-RAGE was higher but not significantly different in diabetic rats compared with normal rats (p = 0.690).
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Biodistribution of 125I-rR-RAGE
In a limited group of animals (three normal rats and two diabetic rats), we studied the distribution of 125I-rR-RAGE at the end of the distribution phase. 125I-rR-RAGE showed a distribution profile between the different organs similar in normal and diabetic rats. Meanwhile, the amount of 125I-rR-RAGE distributed in organs was higher in diabetic rats than in normal rats, which in agreement with the higher distribution volume of 125I-rR-RAGE in diabetic rats (Fig. 6A). The difference between normal and diabetic rats did not reach the statistical level of significance for each organ, probably due to the limited number of animals studied. In normal and diabetic rats, 125I-rR-RAGE distribution was higher in spleen (3501 ± 73 versus 2372 ± 416 ng/g of organ, p = 0.123), liver (1770 ± 31 versus 1454 ± 47 ng/g of organ, p = 0.016), kidney (1636 ± 123 versus 1264 ± 73 ng/g of organ, p = 0.067), and aorta (1414 ± 74 versus 745 ± 121 ng/g of organ, p = 0.027) than in other organs. At the end of the experiment, we did not find any significant difference between normal and diabetic rats. At the end of the distribution phase, the maximum radioactivity corresponding to 125I-rR-RAGE was found in the spleen, and after 96 hr, most of the radioactivity was in the kidney and, to a lesser extent, the liver (Fig. 6B). After intravenous or intraperitoneal administration, distribution of 125I-rR-RAGE in organs was similar at the end of the experiment except that after intraperitoneal administration 125I-rR-RAGE was 50% less tissue distributed.
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Identification of 125I-rR-RAGE
Immunoidentification of 125I-rR-RAGE. After rR-RAGE labeling, >95% of the radioactivity was recovered after TCA precipitation. To further assess whether the radioactivity corresponded to rR-RAGE, we performed immunoprecipitation studies. The specific anti-RAGE antibodies precipitated 75-80% of the radioactivity measured after TCA precipitation in the samples collected after injection in animals. Two hours after injection of 125I-rR-RAGE, 93% of the radioactivity was immunoreactive with the anti-RAGE antibodies (1380 ± 50 versus 1480 ± 20 cpm). From the results, we can conclude that the radioactivity corresponded to rR-RAGE and not to another protein of a similar molecular mass.
SDS-PAGE analysis. Autoradiography of SDS-PAGE (Fig. 7) of plasma samples after 125I-rR-RAGE administration showed one band of ~35 kDa in normal and diabetic rats and two additional bands of 50 and 60 kDa in plasma of diabetic rats. The 35-kDa band corresponds to 125I-rR-RAGE, and the two compounds of higher molecular masses could be the 125I-rR-RAGE bound to AGEs present in vascular compartments of diabetic rats. In urine samples, only one band of 35 kDa was observed in normal and diabetic rats.
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Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Diabetic and normal rats that had received RBCs from syngenic diabetic rats developed a vascular hyperpermeability to albumin. In both animal models, the vascular hyperpermeability was corrected by the infusion of rR-RAGE, as previously shown with purified sRAGE (7). These experiments showed that the effect on the hyperpermeability was specific of RAGE and not due to contaminants in the preparation of RAGE because such contaminants were likely to be different in the preparations of RAGE from bovine lung extracts and of recombinant RAGE from insect cell culture media. The effect of rR-RAGE on vascular hyperpermeability further supports the importance of binding of AGEs to RAGE as a determinant factor of vascular dysfunction in diabetes. Furthermore, using bovine ECs in culture to study endothelial barrier function, we previously showed that RBCs from diabetic patients could provoke an increased albumin or inulin transfer. This effect was prevented by blocking the RAGE-AGE interaction by sRAGE or incubating the AGE RBCs with sRAGE (7). To investigate in detail the effect of sRAGE on the albumin transport across endothelium in rat models, we cloned and expressed rR-RAGE. The sequence of RAGE was found to be highly conserved between species. Using purified rR-RAGE instead of sRAGE from the lung extracts, similar results were obtained, indicating that the observed effect of RAGE on permeability is not due to contaminants in the preparations of RAGE because such contaminants would likely be different in the preparations of RAGE from lung extracts and recombinant RAGE from insect cell culture media. Furthermore, rR-RAGE has kept the functional properties of the purified soluble form and blocked the alteration of EC function by AGEs present on diabetic RBCs.
After intravenous administration in normal and streptozotocin-induced diabetic rats, 125I-rR-RAGE had longer distribution and elimination half-lives than 125I-sRAGE previously studied (7). Two hypotheses could explain the differences between 125I-rR-RAGE and 125I-sRAGE pharmacokinetics. First, the exploration time of 54 hr in the previous study was not sufficient to find a three-compartment model as in the present study, and this drawback could explain the longer distribution and elimination half-lives of 125I-rR-RAGE. Second, although there is little difference in amino acid contents (90% of homology) (4), we do not exclude that clearance of 125I-sRAGE purified from bovine lung was higher than that of 125I-rR-RAGE because of its bovine origin. Furthermore, as we previously observed with 125I-sRAGE (7), distribution and elimination half-lives of 125I-rR-RAGE were longer in diabetic rats than in normal rats, especially the second distribution half-life in diabetic rats (4.01 ± 0.87 hr). The presence of AGEs probably modifies the pharmacokinetics of 125I-rR-RAGE in diabetic rats.
The volume of distribution of 125I-rR-RAGE in normal rats was greater than the total body water (0.7 liter/kg), suggesting a rapid diffusion in tissues. The distribution volume of 125I-rR-RAGE was 2-fold larger in diabetic rats than in normal rats and larger than the volume of the extracellular fluid in rats. Moreover, the duration of the process was considerably longer in diabetic rats (4.01 ± 0.87 hr) than in normal rats (0.21 ± 0.07 hr). This suggests that 125I-rR-RAGE had an additional distribution compartment in diabetic rats. The higher volume of distribution of 125I-rR-RAGE in diabetic rats than in normal rats was apparently not related to the vascular hyperpermeability due to diabetes because murine Fab, a 45-kDa protein that also belongs to the immunoglobulin superfamily, did had have a higher steady state volume of distribution in diabetic rats than in normal rats (0.12 ± 0.01 and 0.19 ± 0.01 liter/kg, respectively; p = 0.006). The more elevated volume of distribution of 125I-rR-RAGE in diabetic rats can be explained by the interaction of 125I-rR-RAGE with AGEs formed on diabetic RBCs (6) and on extracellular proteins. This could constitute an additional compartment of distribution for 125I-rR-RAGE in diabetic rats. Furthermore, the concentration of 125I-rR-RAGE measured at the end of the distribution phase in the different organs was increased in all organs of the diabetic rats compared with normal rats, and this difference was no longer observed when the tissue content was determined 96 hr after 125I-rR-RAGE injection. These results were in agreement with the hypothesis that AGE formation in diabetic rats involves a higher organ distribution and leads to an enhanced volume of distribution in diabetic rats.
Study of the CLR of 125I-rR-RAGE showed that CLR accounted for only 2% of the CL, suggesting that 125I-rR-RAGE was extensively reabsorbed at the kidney level and that peptidic hydrolysis, which characterizes the activity of tubular proximal cells for reabsorbed proteins (17), probably was an important source of 125I-rR-RAGE catabolism. The same analysis of CLR showed that the reabsorption process was less extensive in diabetic than in normal rats. The plasma threshold of the tubular reabsorption was more elevated in two diabetic rats and not observed in the three other diabetic rats. Nevertheless, the study of the urinary excretion rate versus plasma concentration showed that diabetic pathology influenced the urinary excretion of 125I-rR-RAGE.
SDS-PAGE of diabetic rat plasma revealed 125I-rR-RAGE complexes of ~60 kDa, a molecular mass that may correspond to 125I-rR-RAGE/AGE complexes. The molecular mass of the possible 125I-rR-RAGE/AGE complexes is above the glomerular filtration threshold, which is consistent with the fact that the precipitation of 125I-rR-RAGE/AGE complexes with anti-AGE antibodies showed that only a small proportion (0.3-1%) of 125I-rR-RAGE in urine was bound to AGEs. This could corresponds to 125I-rR-RAGE/AGE complexes eliminated in urine, but it could be also underestimated, AGE epitopes recognized by AGE antibodies hidden in the 125I-rR-RAGE/AGE complex formation. These results shown that 125I-rR-RAGE/AGE complexes were not extensively eliminated in urine. We observed in diabetic animals that a higher proportion of 125I-rR-RAGE was distributed in spleen and liver 96 hr after the 125I-rR-RAGE injection, so this could indicate an elimination of 125I-rR-RAGE/AGE complexes via these organs, but this remains to be demonstrated through special designed experiments using metabolically labeled AGEs.
In diabetic rats, pharmacokinetics of 125I-rR-RAGE after intravenous or intraperitoneal administration were characterized by a very slow distribution phase (4.01 ± 1.16 and 3.38 ± 1.16 hr, respectively) and a similar elimination phase (57.17 ± 11.62 and 51.98 ± 6.31 hr, respectively), which confirms that AGEs influence 125I-rR-RAGE pharmacokinetics in both routes of administration. The rate of resorption was quite rapid (Tmax observed in the range 1-2 hr), but the extent was 2-fold less in diabetic rats (35%) than in normal rats (61%). This difference can be explained by the trapping of 125I-rR-RAGE by AGE proteins in the peritoneal cavitae. In diabetic animals, the interaction between endogenous RAGE and AGEs leads to an oxidant stress assessed by the TBARS formation and blocked by administration of sRAGE (6). After intraperitoneal administration, rR-RAGE corrected plasma TBARS similarly to that obtained after intravenous injection, indicating that the absorbed fraction of rR-RAGE after intraperitoneal administration was biologically active.
rR-RAGE present in the vascular compartment could bind with AGEs present on proteins and lipoproteins and inhibit the binding to endogenous RAGE present on ECs. Consequently, it would block the transfer of AGE protein mediated by RAGE/lactoferrin-like receptor complexes through the endothelium (18).
In experimental or human pathologies, several attempts have been made
to prevent ligand/receptor interactions, including the use of
recombinant CD4 in human immunodeficiency virus infections (19) and of
recombinant interleukin-1 receptor in rat autoimmune encephalomyelitis
(20). Because RAGE seems to be a receptor not only for AGEs but also
for amphoterin (21) and amyloid- peptide (22), there is an interest
in better understanding the pathophysiology of RAGE.
The use of recombinant RAGE as well as transgenic animals that overexpress or are deficient in RAGE will allow better delineation of what can be expected from the prevention of binding to endogenous RAGE.
| |
Footnotes |
|---|
Received December 11, 1996; Accepted March 26, 1997
This work was supported by a Research Fellowship (C. R.) provided by Naturalia and Biologia (Paris, France) and a North Atlantic Treaty Organization grant (J.L.W.).
Send reprint requests to: Dr. Jean-Luc Wautier, I.N.T.S., 6, rue Alexandre Cabanel, 75739 Paris Cedex 15, France.
| |
Abbreviations |
|---|
AGE, advanced glycation end product;
EC, endothelial cell;
RAGE, receptor for AGE;
sRAGE, soluble form of RAGE;
RBC, red blood cell;
rR-RAGE, recombinant rat RAGE;
TBARS, thiobarbituric acid-reactive substances;
PBS, phosphate-buffered
saline;
SDS, sodium dodecyl sulfate;
PAGE, polyacrylamide gel
electrophoresis;
Fab, fragment antigen binding;
TBIR, tissue-blood
isotope ratio;
TCA, trichloroacetic acid;
t1/21, first distribution half-life;
t1/22, second distribution half-life;
t1/2z, elimination half-life;
CL, total
clearance;
Vz, volume of distribution of the elimination
phase;
CLR, renal clearance;
Sf9, Spodoptera frugiperda.
| |
References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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1, where
125I/51Cr is the ratio of radioactivity in
tissue versus that in arterial blood sample harvested before excision
of the heart. B, Effect of a transfusion of diabetic RBCs on vascular
permeability. Normal (seven rats), diabetic RBCs (seven rats), or
diabetic RBCs plus rR-RAGE (60 µg/ml) (seven rats) were injected in
normal rats, and TBIR was determined 1 hr after the injection. The
results are presented as mean values. Bars, mean ± standard error. *, p < 0.05. **,
p < 0.01. ***, p < 0.001.
