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Regular Articles |
From the Divisions of Nephrology*
and Clinical
Pharmacology,
Medizinische Hochschule,
Hannover, Germany, and NeXstar
Pharmaceuticals,
Boulder, Colorado
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A large variety of progressive renal diseases is characterized by glomerular mesangial cell proliferation and matrix accumulation.5 PDGF B-chain appears to have a central role in driving both of these processes given that 1) mesangial cells produce PDGF in vitro and various growth factors induce mesangial cell proliferation via induction of auto- or paracrine PDGF B-chain excretion, 2) PDGF B-chain and its receptor are overexpressed in many glomerular diseases, 3) infusion of PDGF-BB or glomerular transfection with a PDGF B-chain cDNA can induce selective mesangial cell proliferation and matrix accumulation in vivo, and 4) PDGF B-chain or ß-receptor knock-out mice fail to develop a mesangium (reviewed in Ref. 4 ).
So far only one study has examined the effect of inhibition of PDGF B-chain in renal disease; Johnson et al, using a neutralizing polyclonal antibody to PDGF, were able to reduce mesangial cell proliferation and matrix accumulation in a rat model of mesangioproliferative glomerulonephritis.6 In this model, injection of an anti-mesangial cell antibody (anti-Thy-1.1) results in complement-dependent lysis of the mesangial cells, followed by an overshooting reparative phase that resembles human mesangioproliferative nephritis.7 Limitations of the study of Johnson et al6 included the necessity to administer large amounts of heterologous IgG and a limitation of the study duration to 4 days due to concerns that the heterologous IgG might elicit a humoral immune reaction. In the present study we have therefore used the anti-Thy-1.1 nephritis model to evaluate the feasibility and efficacy of inhibiting PDGF B-chain in vivo with high-affinity DNA-based aptamers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All aptamers and their sequence-scrambled controls were synthesized by the solid-phase phosphoramidite method on controlled pore glass using an 8800 Milligen DNA synthesizer and deprotected using ammonium hydroxide at 55°C for 16 hours. 2-Fluoropyrimidine nucleoside phosphoramidites were obtained from JBL Scientific (San Luis Obispo, CA). 2'-O-Methylpurine phosphoramidites were obtained from PerSeptive Biosystems (Boston, MA). Hexaethylene glycol (18-atom) spacer was obtained from Glen Research (Sterling, VA). All other nucleoside phosphoramidites were from PerSeptive Biosystems. To prolong the in vivo half-time of the aptamers in plasma, they were coupled to 40-kd polyethylene glycol (PEG). The covalent coupling of PEG to the aptamer (or to its sequence-scrambled control) was accomplished by treating 40-kd PEG N-hydroxysuccinimide ester (Shearwater Polymers, Huntsville, AL) with a primary amine group introduced at the 5' end of the aptamer using trifluoroacetyl-protected pentylamine phosphoramidite. All PEG-aptamer conjugates were purified by anion exchange followed by reverse-phase high-pressure liquid chromatography (HPLC). The binding affinities of various aptamers for PDGF-AB or -BB (R&D Systems, Minneapolis, MN) were determined by the nitrocellulose filter binding method8 or by the competition electrophoresis mobility shift assay.9
Cloning and Expression of Rat PDGF-BB
Rat PDGF-BB for cross-reactivity binding experiments was derived from Escherichia coli transfected with sCR-Script Amp SK(+) plasmid containing the rat PDGF-BB sequence. Rat PDGF-BB sequence was derived from rat lung poly A+ RNA (Clontech, San Diego, CA) through reverse transcription polymerase chain reaction (RT-PCR) using primers that amplify sequence encoding the mature form of PDGF-BB. Rat PDGF-BB protein expression and purification was performed at R&D Systems.
Stability of Aptamers in Rat Plasma in Vitro and in Vivo
The stabilities of DNA-based aptamers in vitro were examined in rat serum at 37°C. Serum was obtained from a Sprague-Dawley rat and was filtered through a 0.45-µm cellulose acetate filter and buffered with 20 mmol/L sodium phosphate buffer. Test ligands were added to the serum at the final concentration of 500 nmol/L. The final serum concentration was 85% as a result of the addition of buffer and aptamer. From the original 900-µl incubation mixture, 100-µl aliquots were withdrawn at various time points and added to 10 µl of 500 mmol/L EDTA (pH 8.0), mixed and frozen on dry ice, and stored at -20°C until the end of the experiment. The amount of full-length oligonucleotide ligand remaining at each of the time points was quantitated by HPLC analysis. To prepare the samples for HPLC injections, 200 µl of a mixture of 30% formamide, 70% 25 mmol/L Tris buffer (pH 8.0) containing 1% acetonitrile was added to 100 µl of thawed time point samples, mixed for 5 seconds, and spun for 20 minutes at 14,000 rpm in an Eppendorf microcentrifuge. The analysis was performed using an anion exchange chromatography column (NuceoPac, Dionex, PA-100, 4 x 50 mm) applying a LiCl gradient. The amount of full-length oligonucleotide remaining at each time point was determined from the peak areas.
Pharmacokinetics of the Modified PDGF Aptamer Conjugated to 40-kd PEG in Vivo
The pharmacokinetic properties of the modified PDGF aptamer
conjugated to 40-kd PEG were determined in Sprague-Dawley rats. Before
animal dosing, the aptamer was diluted with sterile PBS from a stock
solution (also in sterile PBS), to final concentrations between 1 and 2
mg/ml (based on oligonucleotide molecular weight and the ultraviolet
absorption at 260 nm with an extinction coefficient of 0.037 per mg
oligo/ml). A single dose of the aptamer was administered to three
Sprague-Dawley rats by i.v. bolus injection through the tail vein.
Blood samples (approximately 400 µl) were obtained by venipuncture
under isofluorane anesthesia and placed in EDTA-containing tubes. The
EDTA blood samples were immediately processed by centrifugation to
attain plasma and stored frozen at 
-20°C. Time points for blood
sample collection ranged from 2 to 480 minutes
To prepare plasma samples for HPLC analysis, 200 µl of methanol was added to 100 µl of plasma sample, mixed for 30 seconds, and spun in a centrifuge for 10 minutes at 15,000 x g. All of the supernatant was transferred to a new tube and dried under vacuum. Samples were resuspended by addition of 100 µl of 50% formamide and 4% perchloric acid, mixed, and spun as described above. Ninety microliters of supernatant was transferred to a 250-µl limited-volume insert vial for HPLC analysis. Samples were analyzed by anion exchange HPLC (DNAPac PA-100, 4 x 50 mm) with LiCl gradient elution in 30% formamide and monitoring of ultraviolet absorption at 270 nm. Aptamer concentrations were determined based on peak area from a standard curve of the PEG-aptamer conjugate. The HPLC-based analysis described here and the double hybridization analysis described previously9 have produced comparable results for a similar aptamer conjugated to 40-kd PEG (specific binding to vascular endothelial growth factor, Stanley C. Gill, unpublished observations), suggesting that the HPLC analysis indicates the levels of undegraded (full-length) aptamer. Nevertheless, as the PEG moiety has a strong influence on the HPLC retention times of the PEG-aptamer conjugate, we have not at this time ruled out the possibility that some of the partially degraded aptamer may migrate with a similar retention time as the full-length aptamer.
Mesangial Cell Culture Experiments
Human and rat mesangial cells were established in culture, characterized, and maintained as described previously.10 To examine the antiproliferative effect of the aptamers on the cultured mesangial cells, cells were seeded in 96-well plates (Nunc, Wiesbaden, Germany) and grown to subconfluency. They were then growth-arrested for 48 hours in MCDB 302 medium (Sigma, Deisenhofen, Germany) (human mesangial cells) or RPMI 1640 with 1% bovine serum albumin (rat mesangial cells). After 48 hours, various stimuli together with PDGF B-chain aptamer or sequence-scrambled aptamer were added: medium alone, human recombinant PDGF-AA, -AB, or -BB (kindly provided by J. Hoppe, University of Würzburg, Germany), human recombinant epidermal growth factor (EGF; Calbiochem, Bad Soden, Germany), or recombinant human fibroblast growth factor-2 (kindly provided by Synergen/Amgen, Boulder, CO). DNA synthesis in the rat mesangial cells was determined by [3H]thymidine incorporation as described11 after 24 hours of stimulation (of which the last 4 hours were in the presence of [3H]thymidine). In the case of human mesangial cells, after 72 hours of incubation, numbers of viable cells were determined using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT; Sigma) as described.12
Experimental Design
All animal experiments were approved by the local review boards. Anti-Thy-1.1 mesangial proliferative glomerulonephritis was induced in 33 male Wistar rats (Charles River, Sulzfeld, Germany) weighing 150 to 160 g by injection of 1 mg/kg monoclonal anti-Thy-1.1 antibody (clone OX-7; European Collection of Animal Cell Cultures, Salisbury, UK). Rats were treated with aptamers or PEG (see below) from days 3 to 8 after disease induction. Treatment consisted of twice-daily i.v. bolus injections of the substances dissolved in 400 µl of PBS, pH 7.4, for a total of 12 injections. The treatment duration was chosen to treat rats from ~1 day after the onset to the peak of mesangial cell proliferation, which in the OX-7-induced anti-Thy-1.1 nephritis occurs between days 6 and 9 after disease induction. Four groups of rats were studied: 1) 9 rats that received a total of 4 mg (0.33 mg/injection) of the PDGF-B aptamer (coupled to 15.7 mg 40 kd PEG), 2) 10 rats that received an equivalent amount of PEG-coupled, scrambled aptamer, 3) 8 rats that received an equivalent amount (15.7 mg) of 40-kd PEG alone, and 4) 6 rats that received 400-µl bolus injections of PBS alone. Renal biopsies for histological evaluation were obtained on day 6 by intravital biopsy and postmortem on day 9 after disease induction. For intravital biopsies the left kidney was exposed by a flank incision under general anesthesia. A 3- to 4-mm slice was then cut off the lower pole, and bleeding was stopped immediately by gently applying a collagen sponge, followed by wound closure. As judged from serum creatinines, this biopsy technique does not disturb renal function (unpublished observations). Twenty-four-hour urine collections were performed from days 5 to 6 and 8 to 9 after disease induction. The thymidine analogue 5-bromo-2'-deoxyuridine (BrdU; Sigma; 100 mg/kg body weight) was injected intraperitoneally at 4 hours before sacrifice on day 9.
Normal ranges of proteinuria and renal histological parameters (see below) were established in 10 nonmanipulated Wistar rats of similar age.
Renal Morphology
Tissue for light microscopy and immunoperoxidase staining was fixed in methyl Carnoy's solution13 and embedded in paraffin. Four-micron sections were stained with the periodic acid Schiff (PAS) reagent and counterstained with hematoxylin. In the PAS-stained sections the number of mitoses within 100 glomerular tufts was determined.
Immunoperoxidase Staining
Four-micron sections of methyl Carnoy's-fixed biopsy tissue were
processed by an indirect immunoperoxidase technique as
described.13
Primary antibodies were identical to those
described previously14,15
and included a murine monoclonal
antibody (clone 1A4) to 
-smooth muscle actin; a murine monoclonal
antibody (clone PGF-007) to PDGF B-chain; a murine monoclonal IgG
antibody (clone ED1) to a cytoplasmic antigen present in monocytes,
macrophages, and dendritic cells; affinity-purified polyclonal goat
anti-human/bovine type IV collagen IgG preabsorbed with rat
erythrocytes; an affinity-purified IgG fraction of a polyclonal rabbit
anti-rat fibronectin antibody; plus appropriate negative controls as
described previously.14,15
Evaluation of all slides was
performed by an observer who was unaware of the origin of the slides.
To obtain mean numbers of infiltrating leukocytes in glomeruli, more
than 50 consecutive cross sections of glomeruli were evaluated and mean
values per kidney were calculated. For the evaluation of the
immunoperoxidase stains for -smooth muscle actin and PDGF B-chain,
each glomerular area was graded semiquantitatively, and the mean score
per biopsy was calculated. Each score reflects mainly changes in the
extent rather than intensity of staining and depends on the percentage
of the glomerular tuft area showing focally enhanced positive staining:
I, 0% to 25%; II, 25% to 50%; III, 50% to 75%; IV, >75%. We
have recently described that data obtained using this scoring system
are highly correlated with those obtained by computerized
morphometry.16,17
Immunohistochemical Double Staining
Double immunostaining for the identification of the type of
proliferating cells was performed as reported
previously16,17
by first staining the sections for
proliferating cells with a murine monoclonal antibody (clone BU-1)
against bromodeoxyuridine-containing nuclease in Tris-buffered saline
(Amersham, Braunschweig, Germany) using an indirect immunoperoxidase
procedure. Sections were then incubated with the IgG1
monoclonal antibodies 1A4 against -smooth muscle actin and ED1
against monocytes/macrophages. Cells were identified as proliferating
mesangial cells or monocytes/macrophages if they showed positive
nuclear staining for BrdU and if the nucleus was completely surrounded
by cytoplasm positive for -smooth muscle actin or ED1 antigen.
Negative controls included omission of either of the primary
antibodies, in which case no double staining was noted.
In Situ Hybridization for Type IV Collagen mRNA
In situ hybridization was performed on 4-µm sections of biopsy tissue fixed in buffered 10% formalin using a digoxigenin-labeled antisense RNA probe for type IV collagen18 as described.14 Detection of the RNA probe was performed with an alkaline-phosphatase-coupled anti-digoxigenin antibody (Genius nonradioactive nucleic acid detection kit, Boehringer-Mannheim, Mannheim, Germany) with subsequent color development. Controls consisted of hybridization with a sense probe to matched serial sections by hybridization of the antisense probe to tissue sections that had been incubated with RNAse A before hybridization or by deletion of the probe, antibody, or color solution.15 Glomerular mRNA expression was semiquantitatively assessed using the scoring system described above.
Miscellaneous Measurements
Urinary protein was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany) and bovine serum albumin (Sigma) as a standard.
Statistical Analysis
All values are expressed as means ± SD. Statistical significance (defined as P < 0.05) was evaluated using Student t-tests or analysis of variance and Bonferroni t-tests.
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Results |
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High-affinity DNA aptamers to the PDGF B-chain were identified
previously by the SELEX process.8
The consensus secondary
structure motif of these aptamers is a three-way helix junction with a
conserved single-stranded loop at the point of strand exchange (Figure 1A)
. To improve nuclease resistance of
one of the minimal aptamers (a truncated version of aptamer 36t in Ref. 8
), we have synthesized and tested a series of 2'-O-methyl-
or 2'-fluoro-substituted aptamers to identify positions that tolerate
such substitutions without a loss of binding affinity. In addition to
identifying a pattern of allowed 2'-O-methyl- and 2'-fluoro
substitutions, we found that trinucleotide loops on helices II and III
in the aptamer could be replaced with hexaethylene glycol (18-atom)
non-nucleotide spacers without compromising high-affinity binding to
PDGF-AB or -BB (Figure 1A)
. This finding is in agreement with the
notion that the helix junction domain of the aptamer represents the
core of the structural motif required for high-affinity
binding.8
In practical terms, the replacement of six
nucleotides with two spacers is advantageous in that it reduces by four
the number of coupling steps required for the synthesis of the aptamer.
Another support for the importance of helix junction domain in binding
comes from the control aptamer, in which eight nucleotides in the helix
junction region were interchanged without formally changing the
consensus secondary structure (Figure 1A)
. The binding affinity of this
scrambled aptamer for PDGF-BB (Kd 
1
µmol/L) is 10,000-fold lower compared with the binding affinity of
the aptamer used in the experiments described below
(Kd 0.1 nmol/L).
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).
The observed increase in nuclease resistance is in agreement with
previous studies with 2'-substituted nucleic acids.19,20
In
addition to the modifications mentioned above, for all experiments
reported here, the modified DNA aptamer was conjugated to 40-kd PEG.
Importantly, the addition of the PEG moiety to the 5' end of the
aptamer has no effect on the binding affinity of the aptamer for
PDGF-BB (Kd 0.1 nmol/L). Pharmacokinetics of the Modified PDGF Aptamer Conjugated to 40-kd PEG in Vivo
The concentration of the modified PDGF aptamer conjugated to 40-kd
PEG in rat plasma after i.v. injection (1 mg/kg) is shown in Figure 1C
.
The clearance of the aptamer-PEG conjugate from plasma is biphasic with
approximately 47% of the compound being cleared with a half-life of
32.4 ± 13 minutes and 53% of the compound being cleared with a
half-life of 134.5 ± 13 minutes. The concentration of the aptamer
in rat plasma after the i.v. injection is 16 µg/ml (1.6 µmol/L) at
t = 0 and 0.21 µg/ml (21 nmol/L) at t
= 12 hours (extrapolated). Thus, to a first approximation and assuming
a linear increase in plasma concentration of the aptamer at the higher
injected dose in the experiments described below (2.1 to 2.2 mg/kg i.v.
every 12 hours), the aptamer concentration during the treatment period
was not lower than 40 nmol/L (ie, 0.4 µg/ml).
Cross-Reactivity of Aptamers for Rat PDGF-BB
The sequence of PDGF is highly conserved among species, and human
and rat PDGF B-chain sequences are 89% identical.21,22
Nevertheless, in view of the high specificity of
aptamers,23
the correct interpretation of the in
vivo experiments requires understanding of the binding properties
of the aptamers to rat PDGF B-chain. We have therefore cloned and
expressed the mature form of rat PDGF-BB in E. coli. Based
on nitrocellulose filter binding experiments, the PDGF aptamers bound
to rat and human recombinant PDGF-BB with the same affinity
(Kd 0.1 nmol/L).
PDGF B-Chain DNA-Aptamer Specifically Inhibits PDGF-BB-Induced Rat and Human Mesangial Cell Proliferation in Vitro
In growth-arrested rat mesangial cells, the effects of the PDGF-B
aptamer or the scrambled aptamer on mitogen-induced proliferation were
tested. Stimulated growth rates of the cells were not affected by the
addition of scrambled aptamer (Figure 2)
.
At a concentration of 1 µg/ml the PDGF-B aptamer completely inhibited
the PDGF-BB- and -AA-induced growth but had no effect on EGF- or
FGF-2-induced growth (Figure 2)
. In an additional experiment we also
documented a 84% inhibition of the mesangial cell growth induced by 50
ng/ml PDGF-BB at an aptamer concentration of 0.5 µg/ml, ie, the
minimal aptamer level achieved in vivo (see above).
Inhibition of PDGF-BB-induced growth by the PDGF-B aptamer was not due
to cell death as evidenced by trypan blue exclusion of the cells.
Furthermore, using morphological criteria, in particular, nuclear
condensation, no evidence for mesangial cell apoptosis was noted under
these conditions.
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, the PDGF-B
aptamer also completely inhibited PDGF-BB-induced human mesangial cell
growth. PDGF-AB- and -AA-induced mesangial cell growth also tended to
be lower with the PDGF-B aptamer, but these differences failed to reach
statistical significance. In contrast, no effects of the PDGF-B aptamer
on either EGF- or FGF-2-induced growth were noted (Table 1)
. Similar
effects were noted if the aptamers were used at a concentration of 10
µg/ml (data not shown).
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After the injection of anti-Thy-1.1 antibody, PBS-treated animals developed the typical course of the nephritis, which is characterized by early mesangiolysis and followed by a phase of mesangial cell proliferation and matrix accumulation on days 6 and 9. No obvious adverse effects were noted after the repeated injection of aptamers or PEG alone, and all rats survived and appeared normal until the end of the study.
In PAS-stained renal sections the mesangioproliferative changes on days
6 and 9 after disease induction were severe and indistinguishable among
rats receiving PBS, PEG alone, or the scrambled aptamer (Figure 3
, A to C). Histological changes were
markedly reduced in the PDGF-B aptamer-treated group (Figure 3D)
. To
evaluate the mesangioproliferative changes, various parameters were
analyzed.
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Glomerular cell proliferation, as assessed by counting the number
of glomerular mitoses, was not significantly different between the
three control groups on days 6 and 9 (Figure 4)
. Compared with rats receiving the
scrambled aptamer, treatment with PDGF-B aptamer led to a reduction of
glomerular mitoses by 64% on day 6 and by 78% on day 9 (Figure 4)
. To
assess the treatment effects on mesangial cells, we immunostained the
renal sections for -smooth muscle actin, which is expressed by
activated mesangial cells only.24
Again, there were no
significant differences between the three control groups on days 6 and
9. However, the immunostaining scores of -smooth muscle actin
were significantly reduced on days 6 and 9 in the PDGF-B
aptamer-treated group (Figure 4)
. To specifically determine whether
mesangial cell proliferation was reduced, we double immuno-stained
PDGF-B aptamer-treated rats and scrambled aptamer-treated rats for a
cell proliferation marker (BrdU) and -smooth muscle actin. The data
confirmed a marked decrease of proliferating mesangial cells on day 9
after disease induction: 2.2 ± 0.8
BrdU-positive/-smooth-muscle-actin-positive cells per 100 glomerular
cross sections in PDGF-B aptamer-treated rats versus
43.3 ± 12.4 cells in rats receiving the scrambled aptamer, ie, a
95% reduction of mesangial cell proliferation (Figure 5)
. In contrast, no effect of the PDGF-B
aptamer was noted on proliferating monocytes/macrophages on day 9 after
disease induction (PDGF-B aptamer-treated rats: 2.8 ± 1.1
BrdU+/ED-1+ cells per 100 glomerular cross
sections; scrambled aptamer-treated rats: 2.7 ± 1.8).
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By immunohistochemistry the glomerular PDGF B-chain expression was
markedly up-regulated in all three control groups (Figure 4)
, similar
to previous observations.15
In the PDGF-B aptamer-treated
group the glomerular overexpression of PDGF B-chain was
significantly reduced in parallel with the reduction of
proliferating mesangial cells (Figure 4)
. This reduction was not due to
masking of the PDGF B-chain epitope recognized by the anti-PDGF B-chain
antibody, as the immunostaining intensity was not affected in renal
sections that had been preincubated with the PDGF-B aptamer
(immunostaining scores in sections preincubated with buffer, 2.70; with
PDGF-B aptamer, 2.97; with scrambled aptamer, 2.78; means of two
experiments each).
Reduction of Glomerular Monocyte/Macrophage Influx
The glomerular monocyte/macrophage influx was significantly
reduced in the PDGF aptamer-treated rats as compared with rats
receiving scrambled aptamer on days 6 and 9 after disease induction
(Figure 4)
.
Effects on Proteinuria
Moderate proteinuria of up to 147 mg/24 hours was present on day 6
after disease induction in the three control groups (Figure 4)
.
Treatment with the PDGF-B aptamer reduced the mean proteinuria on day
6, but this failed to reach statistical significance (Figure 4)
.
Proteinuria on day 9 after disease induction was low and similar in all
four groups (Figure 4)
.
Reduction of Glomerular Matrix Production and Accumulation
By immunohistochemistry, marked glomerular accumulation of type IV
collagen and fibronectin was noted in all three control groups (Figure 6)
. The overexpression of both glomerular
type IV collagen and fibronectin was significantly reduced in PDGF-B
aptamer-treated rats (Figure 6)
. By in situ hybridization,
the decreased glomerular protein expression of type IV collagen in
PDGF-B aptamer-treated rats was shown to be associated with decreased
glomerular synthesis of this collagen type (Figure 6)
. Sense controls
for the in situ hybridizations were negative and similar to
those published recently25
(Figure 6E)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major finding of the present study was that treatment with PDGF-B
aptamer from days 3 to 9 after disease induction resulted in a near
complete arrest of the overshooting mesangial cell proliferation and
thereby led to a marked reduction of glomerular hypercellularity.
Further support for the reduction of mesangial cell proliferation is
provided by the observations that the glomerular de novo
expression of -smooth muscle actin and the overexpression of PDGF
B-chain were also significantly inhibited by the PDGF-B aptamer. Both
of these proteins are selectively up-regulated in activated mesangial
cells in the anti-Thy-1.1 model,15,24
and consequently, any
intervention that reduces mesangial cell hyperplasia should also reduce
expression of these proteins. It is of interest to note that mesangial
cell proliferation as assessed by double immunostaining for BrdU and
-smooth muscle actin expression was reduced by 95% although
glomerular mitoses were reduced by only 78% on day 9. This discrepancy
suggests that proliferation of other glomerular cells may
be independent of PDGF (as demonstrated, for example, by the
determination of proliferating monocytes/macrophages) and lends support
to the highly specific action of the PDGF-B aptamer.
In addition to selectively abrogating mesangial cell proliferation, treatment with the PDGF-B aptamer also reduced the glomerular monocyte/macrophage influx and accumulation of extracellular matrix. Both of these latter processes are known to depend to a large degree on other cytokines produced by activated mesangial cells: glomerular macrophage influx on the production of monocyte chemoattractant protein-128 and matrix overproduction on the release of transforming growth factor-ß.29 The present data therefore suggest that PDGF B-chain is either linked to the mesangial cell production of these latter cytokines, as indicated by cell culture studies,30 or that PDGF B-chain is directly and independently involved in chemotaxis and matrix production. Regardless of these issues, the present study establishes the release of PDGF B-chain as a central pathogenetic event in the development of mesangioproliferative glomerulonephritis. Our data also suggest that macrophage- or platelet-derived PDGF is unlikely to play a major role in the mediation of mesangioproliferative changes in this model, as influx of both cell types peaks at ~24 hours,13 ie, 2 days before the initiation of anti-PDGF treatment.
Similar, albeit quantitatively somewhat lower, effects were also noted
in our previous study with a neutralizing antibody to PDGF B-chain in
the anti-Thy-1.1 nephritis model; treatment resulted in a 57%
reduction of glomerular cell proliferation on day 4 and reduced
glomerular matrix accumulation by approximately one score
point.6
One important concern in that study was that large
amounts of IgG (600 mg/kg/day), both neutralizing anti-PDGF antibody
and control IgG, had to be administered on a daily basis to the rats.
Large doses of IgG can ameliorate the course of immune-mediated renal
disease,31
and the therapeutic effect of IgG per
se is variable from batch to batch (R. J. Johnson and
J. Floege, personal observation). Second, mesangial cells express
Fc-
receptors and modulate their cytokine release on binding of IgG
to Fc receptors.10
These observations, in addition to the
immunogenicity of heterologous antibodies, impose considerable
restrictions on the interpretation of such studies.
At present, no other specific PDGF B-chain antagonists have been tested in vivo. Trapidil, an anti-platelet agent, has been shown to interfere with PDGF B-chain binding to mesangial PDGF receptors, but its action is not specific for PDGF.32 Preliminary data suggest that a blocker of the PDGF receptor-associated tyrosine kinase may also ameliorate the course of mesangioproliferative nephritis,33 but again, the compound was not specific for PDGF and also reduced, for example, basic-FGF-induced cell proliferation.
The possibility to develop aptamer antagonists for defined biological mediators has become an attractive novel therapeutic approach in various diseases.23 These previous studies, in which thrombin, selectins, and neutrophil elastase were targeted, have demonstrated the ability of aptamers to act as antagonists of plasma or leukocyte proteins in vivo.34-36 The present study is the first to demonstrate that specific antagonism of peptide growth factors, which are produced in a spatially much more confined manner, by high-affinity aptamers in vivo is not only feasible but also highly effective. In pilot studies the high effectiveness of the PDGF-B aptamer was also demonstrated in an experiment in which the injection of 5 mg of the aptamer at a single time point, ie, day 3 of the nephritis, was as efficient as several days of twice-daily treatment with a total of 10 to 30 mg (unpublished data). Furthermore, in pilot experiments we have observed that the effects of the PDGF-B aptamer in vivo are dose dependent and can be demonstrated with doses as low as 2 mg total. In support of our findings, the PDGF aptamer used in this study has recently exhibited efficacy in the rat model of restenosis (C.-H. Heldin and A. Östman, personal communication).
Apart from their specificity and potency, the use of aptamers largely circumvents problems of Fc receptor binding. Furthermore, although the effort to thoroughly examine the immunogenicity of aptamers is still in progress, extensive in vivo data obtained to date suggest that aptamers induce little if any immune response (J. Bill and G. Biesecker, unpublished results). Another attractive feature of aptamers is that their pharmacokinetic behavior can easily be altered, for example, by altering the molecular size (eg, via variation of the length of the attached PEG), hydrophobicity (eg, via packaging them in or on liposomes9 ), or other features. A potential problem with the use of nucleic-acid-based antagonists is their polyanionic character, which might in itself affect the course of diseases. Thus, other polyanionic compounds, for example, heparins or heparan sulfate proteoglycans, are also effective in reducing mesangial cell proliferation in the anti-Thy-1.1 nephritis model.14 However, in the present study, this concern was resolved by the ineffectiveness of a scrambled aptamer, which is identical in composition and predicted secondary structure to the PDGF-B aptamer but whose binding affinity for PDGF B-chain is dramatically lower.
In conclusion, we describe the efficacy of a high-affinity nucleic-acid-based growth factor antagonist in vivo and thereby extend the currently available evidence that PDGF B-chain is a central mediator in mesangioproliferative glomerulonephritis. The specificity and effectiveness demonstrated with the currently used aptamer opens the possibility for growth factor inhibition over prolonged periods. Aptamers may thus be used both to elucidate the role of individual growth factors that represent potential targets for therapeutic intervention and to serve as drug candidates for the treatment of disease states characterized by growth factor overexpression.
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Acknowledgements |
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Footnotes |
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Supported by a grant (SFB 244/C12) and a Heisenberg stipend of the Deutsche Forschungsgemeinschaft to J. Floege.
J. Floege and T. Ostendorf contributed equally to this study.
Accepted for publication October 11, 1998.
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N: Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 1996, 35:14413-14424[Medline]
N: Liposome-anchored vascular endothelial growth factor aptamers. Bioconj Chem 1998, 9:573-582[Medline]
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  B. Venkatesan, N. Ghosh-Choudhury, F. Das, L. Mahimainathan, A. Kamat, B. S. Kasinath, H. E. Abboud, and G. G. Choudhury Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B FASEB J, October 1, 2008; 22(10): 3469 - 3482. [Abstract] [Full Text] [PDF]
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T. Rampino, G. Soccio, M. Gregorini, C. Guidetti, M. Marasa, M. Maggio, V. Panichi, M. Migliori, C. Libetta, and A. Dal Canton Neutralization of Macrophage-Stimulating Protein Ameliorates Renal Injury in Anti-Thy 1 Glomerulonephritis J. Am. Soc. Nephrol., May 1, 2007; 18(5): 1486 - 1496. [Abstract] [Full Text] [PDF]
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T. Ostendorf, S. Rong, P. Boor, S. Wiedemann, U. Kunter, U. Haubold, C. R.C. van Roeyen, F. Eitner, H. Kawachi, G. Starling, et al. Antagonism of PDGF-D by Human Antibody CR002 Prevents Renal Scarring in Experimental Glomerulonephritis J. Am. Soc. Nephrol., April 1, 2006; 17(4): 1054 - 1062. [Abstract] [Full Text] [PDF]
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C. R.C. van Roeyen, F. Eitner, S. Martinkus, S. R. Thieltges, T. Ostendorf, D. Bokemeyer, B. Luscher, J. M. Luscher-Firzlaff, J. Floege, and P. R. Mertens Y-Box Protein 1 Mediates PDGF-B Effects in Mesangioproliferative Glomerular Disease J. Am. Soc. Nephrol., October 1, 2005; 16(10): 2985 - 2996. [Abstract] [Full Text] [PDF]
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T. Ostendorf, C. van Roeyen, R. Westenfeld, A. Gawlik, M. Kitahara, E. de Heer, D. Kerjaschki, J. Floege, and M. Ketteler Inducible Nitric Oxide Synthase-Derived Nitric Oxide Promotes Glomerular Angiogenesis via Upregulation of Vascular Endothelial Growth Factor Receptors J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2307 - 2319. [Abstract] [Full Text] [PDF]
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K. L. Hudkins, D. G. Gilbertson, M. Carling, S. Taneda, S. D. Hughes, M. S. Holdren, T. E. Palmer, S. Topouzis, A. C. Haran, A. L. Feldhaus, et al. Exogenous PDGF-D Is a Potent Mesangial Cell Mitogen and Causes a Severe Mesangial Proliferative Glomerulopathy J. Am. Soc. Nephrol., February 1, 2004; 15(2): 286 - 298. [Abstract] [Full Text] [PDF]
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 S.-L. Lin, R.-H. Chen, Y.-M. Chen, W.-C. Chiang, T.-J. Tsai, and B.-S. Hsieh Pentoxifylline Inhibits Platelet-Derived Growth Factor-Stimulated Cyclin D1 Expression in Mesangial Cells by Blocking Akt Membrane Translocation Mol. Pharmacol., October 1, 2003; 64(4): 811 - 822. [Abstract] [Full Text] [PDF]
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J. Floege, F. Eitner, C. van Roeyen, and T. Ostendorf PDGF-D and Renal Disease: Yet Another One of Those Growth Factors? J. Am. Soc. Nephrol., October 1, 2003; 14(10): 2690 - 2691. [Full Text] [PDF]
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T. Ostendorf, C. R.C. van Roeyen, J. D. Peterson, U. Kunter, F. Eitner, A. J. Hamad, G. Chan, X.-C. Jia, J. Macaluso, G. Gazit-Bornstein, et al. A Fully Human Monoclonal Antibody (CR002) Identifies PDGF-D as a Novel Mediator of Mesangioproliferative Glomerulonephritis J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2237 - 2247. [Abstract] [Full Text] [PDF]
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W. Kriz, B. Hahnel, H. Hosser, T. Ostendorf, S. Gaertner, B. Kranzlin, N. Gretz, F. Shimizu, and J. Floege Pathways to Recovery and Loss of Nephrons in Anti-Thy-1 Nephritis J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1904 - 1926. [Abstract] [Full Text] [PDF]
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 V. Balasubramaniam, T. D. Le Cras, D. D. Ivy, T. R. Grover, J. P. Kinsella, and S. H. Abman Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L826 - L833. [Abstract] [Full Text] [PDF]
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 K. Pietras, K. Rubin, T. Sjoblom, E. Buchdunger, M. Sjoquist, C.-H. Heldin, and A. Ostman Inhibition of PDGF Receptor Signaling in Tumor Stroma Enhances Antitumor Effect of Chemotherapy Cancer Res., October 1, 2002; 62(19): 5476 - 5484. [Abstract] [Full Text] [PDF]
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