Research Group Immunobiology, Heinrich-Heine-University of Düsseldorf, D-40001 Düsseldorf, Germany
Submitted 3 February 2003 ; accepted in final form 21 March 2003
ABSTRACT
The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.
endothelium; inflammation; nitric oxide; inducible nitric oxide synthase
The power of AS-ODN as specific inhibitors of gene expression is strongly limited by their low spontaneous cellular uptake (15). Furthermore, ODN are rapidly degraded by nucleases if not chemically modified and stabilized (17, 19, 27). To circumvent the obstacle of degradation, a variety of improvements, for instance phosphate-modified analogs, have been developed (20). The problem of effective cellular ODN uptake will be solved by using uptake enhancers like cationic lipids (4). These substances ensure nucleotide uptake and release from endosomal compartments (10). Here, medium constituents and incubation times have a marked influence on micelle formation, as well as incorporation and intracellular localization of ODN (25, 26). Furthermore, it is still controversial whether ODN have to accumulate in the nucleus or whether cytoplasmic accumulation will lead to the desired inhibition (24). Because there are numerous parameters requiring consideration, precise experimental protocols for all the factors involved are necessary for successful AS inhibition. In addition, an inflammatory surrounding as created by cytokines may influence the effects of ODN or lipid vehicles, thereby affecting the outcome.
Chronic inflammatory conditions are usually associated with an
overexpression of the inducible nitric oxide synthase (iNOS) as a result of
proinflammatory cytokines. The resulting high-output production of nitric
oxide (NO) is often associated with cell damage or tissue destruction
(1,
3,
19). Because such unregulated
iNOS activity and high concentrations of its product, NO, have been found to
be associated with many diseases
(13), the search for a highly
specific method for iNOS inhibition is continuing. Substrate deprivation,
competitive inhibition by using substrate analogs
(8), manipulation of cofactor
availability, and the manipulation of the expression or functionality of
iNOS-relevant transcription factors like NF-B or I
B
(12) represent some of the
experimental approaches for iNOS inhibition. However, all of these techniques
raise problems due to toxicity or lack of specificity. It has been tried only
once to inhibit iNOS protein expression in endothelium
(22). Similar to other
approaches, unsatisfactory results concerning the level of iNOS inhibition
were reported. It is surprising that endothelial iNOS has only rarely been
chosen for iNOS-directed AS treatment, because endothelial cells (EC)
represent a prime target for in vivo intervention and are inevitably affected
by AS-ODN when applied via the blood. Furthermore, iNOS and its product, NO,
represent a system of major importance in a wide range of endothelial
functions, e.g., oxidative stress, inflammation, antimicrobial reactions, and
regulation of gene expression
(2,
14,
16,
18). In addition, excessive NO
formation is also held responsible for the circulation failure in patients
with septic shock.
It is the aim of the present study to provide an optimized experimental protocol for AS experiments in primary, nontransformed EC, combining low toxicity with strong and specific AS-mediated inhibition.
To examine the effectiveness of transmembrane carriers and to improve the
rate of ODN incorporation, we investigated the three widely used transfection
re-agents FuGENE 6, LipofectAMINE, and Lipofectin and their usefulness under
various conditions concerning choice of medium and incubation times. By means
of fluorescence microscopy, we explored how different conditions and methods
of preparing AS-ODN-lipid complexes determine the intracellular localization
of ODN. In parallel, we investigated the inhibitory effects of AS-ODN on
cytokine-mediated endothelial iNOS mRNA formation and iNOS protein expression,
as well as the specificity of the inhibition achieved by monitoring the
induction and expression of interleukin-1 (IL-1
), driven by the
same cytokines.
MATERIALS AND METHODS
Reagents. Recombinant human IL-1 was from HBT (Leiden,
Netherlands). Recombinant murine interferon-
(IFN-
) and
recombinant murine tumor necrosis factor (TNF-
) were from Genzyme
(Cambridge, MA). Endothelial cell growth supplement (ECGS), LPS (from
Salmonella typhimurium), neutral red (3% solution), type I collagen,
collagenase (from Clostridium histolyticum), rabbit anti-human von
Willebrand Factor (vWF) antiserum, 2-mercaptoethanol, and
anti-
-tubulin-antibody were from Sigma (Deisenhofen, Germany). Rat
endothelium specific monoclonal antibody Ox43 was from Serotec (Camon,
Wiesbaden, Germany), monoclonal anti-mouse endothelial nitric oxide synthase
(eNOS) and anti-mouse iNOS antibodies were from Transduction Laboratories
(Lexington, KT), and peroxidase-conjugated porcine anti-rabbit IgG was from
DAKO (Hamburg, Germany). Peroxidase-conjugated goat anti-mouse IgG was from
Zymed (San Francisco, CA), trypsin, EDTA, and fetal calf serum (FCS, endotoxin
free) were from Boehringer Mannheim (Mannheim, Germany), Omniscript RT kit and
Taq Core PCR kit were from Qiagen (Hilden, Germany), FuGENE 6 was
from Roche (Mannheim, Germany), and RPMI 1640 (endotoxin free),
oligo(dT)15-primer, Lipofectin, LipofectAMINE, and Opti-MEM
serum-reduced medium were from Life Technologies (Eggenstein, Germany).
3,3'-Diaminobenzidine (DAB) was from Serva (Heidelberg, Germany).
Antisense oligonucleotides and controls directed to iNOS have been designed
and manufactured by Biognostik (Göttingen, Germany). Chosen for
inhibition of iNOS were anti-sense oligodesoxynucleotides with or without FITC
label (5'-TTTGCCTTATACTGTTCC-3'). As controls, we used two
oligodesoxynucleotides with identical purine and pyrimidine content
(5'-ACTACTACACTAGACTAC-3' and
5'-ATATCCTTCCAGTACAG-3'), the second one of which was also FITC
labeled.
Cell cultures and cellular characterization. Rat aorta EC were isolated by outgrowth from rat aortic rings exactly as described (21). Briefly, aortic segments were placed on top of a collagen gel (1.8 mg collagen/ml) in 24-well tissue culture plates and incubated in RPMI 1640 with 20% FCS and 100 µg/ml ECGS for 4 to 6 days, depending on the degree of cellular outgrowth. Aortic explants were then removed, and cells were detached with 0.25% collagenase in HBSS and replated onto plastic culture dishes in RPMI 1640/20% FCS. Cells were subcultured for up to 10 passages, and removal from culture dishes for each passage was performed by treatment with 0.05% trypsin/0.02% EDTA in isotonic NaCl for 3 min.
EC were characterized by using a crossreacting rabbit-anti-human-vWF antiserum, the rat vascular endothelium specific monoclonal antibody Ox43, the monoclonal mouse-anti-eNOS antibody, and the respective secondary peroxidase-conjugated porcine anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG antisera at conditions exactly as described previously (21). EC showed the antigen phenotype vWFhigh Ox43high eNOShigh exactly as published (21). The labeling also proved the purity of these cultures, since the respective staining patterns were found in virtually all cells.
Control experiments concerning ODN uptake and intracellular accumulation were also performed with the mouse fibroblast cell line L929, usually maintained in RPMI 1640/10% FCS or as indicated.
Experimental design. All experiments with EC were performed with
cells from passages 2 to 8. EC or L929 were cultured in 6-
(2 x 105 cells) or 12-well (0.81 x
105 cells) tissue culture plates in RPMI 1640 (20% FCS for EC, 10%
FCS for L929) and incubated overnight to allow attachment before supernatant
was replaced by 1) fresh Opti-MEM serum-reduced medium, 2)
RPMI 1640/10% or 20% FCS, or 3) RPMI 1640 without FCS. Cells were
either resident or activated by cytokines (IL-1, TNF-
, and
IFN-
, each at 1,000 U/ml) before analysis by fluorescence
microscopy.
Analysis of oligonucleotide uptake without lipid encapsulation. After overnight attachment in 6- or 12-well plates, culture supernatants were replaced by 1,000 µl (6-well) or 600 µl (12-well) of fresh medium containing the phosphorothioate AS-ODN at concentrations indicated for 2 to 72 h and monitored for positive uptake. Cells were then activated by cytokines, and mRNA formation, protein expression, and nitrite accumulation were measured.
Analysis of ODN uptake with lipid encapsulation. To examine the impact of transmembrane vehicles on ODN incorporation, FuGENE 6, LipofectAMINE, and Lipofectin were investigated as lipid vehicles.
FuGENE 6 was used at amounts of 1.515 µl in 600 µl of Opti-MEM serum-reduced medium on 12-well culture dishes. Diluted ODN, used at concentrations from 0.13 to 0.49 µM, were added to the prediluted FuGENE 6 reagent.
LipofectAMINE was used according to the manufacturer's recommendations at concentrations as indicated. A solution of 1 µl/ml has a final concentration of 1.03 µM 2,3-dioleyloxy-N-[2(sperminecarboxamide]ethyl-N,N-dimethyl-1-propanaminium trifluoroacetate and 0.673 µM dioleoylphosphatidyl ethanolamine (DOPE). Vehicles and ODN were used at concentrations as indicated in 12-well plates with 600 µl of supernatant.
Lipofectin was prepared according to the recommendations of the supplier and used at concentrations as given. A solution of 1 µl/ml has a final concentration of 0.75 µM N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride and 0.68 µM DOPE. Lipofectin was applied in six-well culture dishes with 1 ml of supernatant.
When using LipofectAMINE or Lipofectin, ODN and lipid were diluted separately in Opti-MEM or RPMI 1640/20% FCS, 10% FCS, or 0% FCS, incubated at room temperature for 15 min (LipofectAMINE) or 45 min (Lipofectin), and then combined and incubated at room temperature for 15 min to allow for micelle formation.
Cell treatments. After overnight attachment of freshly seeded cultures in 6- or 12-well plates, supernatants were replaced by 1 ml or 600 µl of fresh Opti-MEM, RPMI 1640/20% or 10% FCS according to the cell type, or RPMI 1640/0% FCS, containing lipid alone or ODN-lipid complexes prepared as described. After incubation periods of 2 to 24 h, cells were activated by cytokine challenge and incubated for an additional 24 h in cytokine-containing Opti-MEM, RPMI 1640/20% or 10% FCS, or RPMI 1640 without FCS.
Additionally, Lipofectin and LipofectAMINE were checked for endotoxin content using the Limulus amoebocyte lysate test, and endotoxin levels were always below the detection limit.
Growth rates of cell cultures and viability. Cell growth was determined by neutral red staining. Cells were incubated for 90 min with neutral red (1:100 dilution of a 3% solution), washed twice with PBS, dried, and lysed with isopropanol containing 0.5% 1 N HCl. Supernatants were measured at 530 nm. Additionally, viability of EC and L929 was routinely controlled using trypan blue exclusion.
Nitrite determination. Cellular NO production was measured by quantifying the nitrite accumulation in culture supernatants of EC by using the diazotization reaction as modified by Wood et al. (23) and NaNO2 as standard.
Reverse transcription and polymerase chain reaction. Total cellular RNA (with 1 µg RNA/probe) was prepared using the Omniscript RT kit, and reverse transcription (RT) was carried out at 37°C for 60 min with oligo (dT) (15 mer) as primer. The cDNA (500 ng each) was used for PCR with primer ODN and amplification protocols as shown in Table 1.
|
PCR products were subjected to electrophoresis on 1.8% agarose gels. Bands were visualized by ethidium bromide staining. Densitometric analysis of the visualized amplification products was performed by using the KODAK 1D software (KODAK, Stuttgart, Germany).
Western blot analysis of the iNOS protein. Cells, treated as
indicated, were washed, scraped from the dishes, lysed, transferred to a
microcentrifuge tube, and boiled for 5 min in electrophoresis buffer. Proteins
(30 µg per lane) were separated by electrophoresis in a 12%
SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Further
incubations were 2 h blocking buffer (2% BSA, 5% nonfat milk powder, 0.1%
Tween 20 in PBS buffer), 1 h at 37°C with a 1:2,000 dilution of the
monoclonal anti-iNOS antibody, and 1 h with a 1:2,000 dilution of the
secondary horseradish peroxidase-conjugated rabbit-anti-mouse-IgG antibody.
Finally, blots were incubated for 5 min in ECL reagent (Pierce, Rockford, IL)
and exposed to an autoradiographic film. To control for equal loading of total
protein in all lanes, blots were also stained with a mouse
anti--tubulin antibody at a dilution of 1:2,000.
Statistical analysis. Data are given as arithmetical means ± SD. Values were calculated using the Student's t-test (two-tailed for independent samples).
RESULTS
ODN uptake in the absence or presence of vehicles. Cultured EC or L929 cells were incubated with various concentrations of ODN in RPMI 1640 with FCS (20% for EC, 10% for L929), FCS-free RPMI 1640, or Opti-MEM serum-reduced medium (Opti-MEM).
In the absence of transport vehicles, fluorochrome-labeled ODN were found dispersed in the supernatant or attached to the outer cell membrane, and intracellular accumulation was never observed, irrespective of the various concentrations, different medium conditions, cytokine activation, or incubation times (Fig. 1, AC). The longer the incubation times and the higher the concentration of FITC-labeled ODN, the brighter was the green fluorescence surrounding the cells and covering the culture dishes.
|
Using the transmembrane vehicle FuGENE 6, a similar picture was seen as in the absence of uptake enhancers. Although ODN and lipid concentrations, as well as incubation times and culture medium conditions (RPMI, Opti-MEM, FCS), were again varied, no uptake of ODN was observed in any combination (Fig. 1D).
With LipofectAMINE (Fig. 1, EH), good intracellular labeling of cells was observed with 4 µl of lipid/600 µl of medium and ODN at a concentration of 0.7 µM. Best uptake of ODN was achieved by leaving lipid and ODN complexes on the cells for at least 4 h before cytokine activation. Earlier removal of ODN caused a significantly lower uptake rate and, in addition, fluorescence microscopy showed ODN still attached to the outer cell membrane and not yet incorporated (Fig. 1E). Incubation of cells for at least 4 h led to accumulation of ODN in the nucleus, as evidenced by bright fluorescence signals over the nuclei with hardly any label in the cytoplasm (Fig. 1F). ODN were still found in the nucleus after an incubation period of 20 h (Fig. 1, G and H).
When using Lipofectin, good labeling of cells was observed with 6 µl of lipid in 1 ml Opti-MEM and ODN concentrations of 0.6 µM (Fig. 1, J and K). Again, most of the label accumulated in the nuclei, but here, a significant portion was also seen in cytoplasmic organelles. After 20 h, these cytoplasmic aggregates had disappeared and left a slight homogenous coloring of the cytoplasma, with the nuclei showing intensive fluorescence signals.
Cell viability. Next, we monitored cell viability under various incubation conditions with or without lipid and with or without ODN (Fig. 2). Incubation conditions with negative uptake in the absence of vehicles showed no toxicity, irrespective of ODN concentrations (Fig. 2A). Unloaded LipofectAMINE vesicles or ODN-loaded micelles showed significant, concentration-dependent toxic effects (Fig. 2, B and C, and Table 2).
|
|
|
Incubation protocols influencing ODN uptake and intracellular ODN localization. Because LipofectAMINE mediated excellent uptake but caused significant toxicity at the same time, we also examined the impact of the presence of FCS on viability and uptake modalities in EC and L929 fibroblasts incubated and cultured under otherwise identical conditions as EC. We find that using FCS during the whole experiment decreases toxicity and does not interfere with uptake but leads to a different intracellular accumulation pattern (see Table 2). As shown in Fig. 3, AD, in the presence of FCS, label accumulates around the nuclear envelope and not within the nuclear compartment. In contrast, incubation in the absence of serum leads to nuclear accumulation (Fig. 3, EH, and Table 2). Lipofectin-mediated ODN uptake also reveals cytoplasmic accumulation without any ODN labels in the nucleus if FCS is present during lipid-ODN complex formation.
|
The ratio of lipid to ODN is an important factor in determining the formation of intact lipid-ODN complexes and, thus, an uptake experiment was performed using fixed ODN concentrations of 0.7 µM and increasing concentrations of LipofectAMINE (Fig. 3, EH). If LipofectAMINE amounts were too low compared with ODN concentrations, no adequate lipid-ODN constructs were formed and FITC-labeled ODN accumulated around cells and in the culture supernatant without any stained nuclei (Fig. 3, J and K). The effect of FCS and the lipid-to-ODN ratio on ODN uptake was identical in L929 fibroblasts and in EC.
Because toxicity was high with LipofectAMINE in both cell types tested (Fig. 2, B and C, and Table 2), we now concentrated on the use of Lipofectin and studied the effect of various incubation times. As shown in Fig. 4, ODN incorporation into the nuclei of EC and L929 increased signifi-cantly between 4 and 5.5 h of incubation from 36.5 ± 21.5% to 88 ± 11% of the cells, although longer incubation periods did not further improve labeling. This strong intracellular accumulation of ODN was achieved in subconfluent cultures only (4060% of confluency), whereas higher cell densities (confluency of 80% or higher) led to decreases in uptake efficiency (data not shown). Again, data did not differ significantly between L929 and EC.
|
|
Impact of AS-ODN on iNOS mRNA synthesis, protein expression, and enzyme
activity. With the conditions described above, we next determined the
effect of ASODN on iNOS mRNA expression in EC cultures incubated for a 5.5
h-period, followed by cytokine activation (IFN-, IL-1
,
TNF-
, 1,000 U/ml each) for 24 h. As seen in
Fig. 5, A and
B, cytokine-mediated activation induced the de novo
formation of iNOS mRNA in sham-treated cells as expected. Addition of AS-ODN
via Lipofectin as vehicle resulted in some minor decrease in iNOS mRNA
expression to 70 ± 10% of the level of sham-treated or control
ODN-treated cells (Fig.
5A, lane 1), whereas Lipofectin with either one
of two control ODN sequences (see Reagents) did not alter the
cytokine-mediated iNOS expression (Fig.
5A, lane 2). Interestingly, the vehicle control,
i.e., empty Lipofectin vesicles, led to a significant and dose-dependent
increase in iNOS-specific mRNA formation
(Fig. 5A, lane
4, and 5B). This lipid-mediated increment in iNOS expression was
completely abolished by loading the vehicle with control ODN
(Fig. 5A, lane
2). Because endotoxin contamination of all lipid formulations was
essentially below the detection limit as measured by Limulus lysate assay,
this iNOS upregulation must be regarded as a lipid-specific effect.
Next, we examined the specificity of this AS-mediated effect by monitoring
the impact on another gene, also expressed de novo in the presence of
proinflammatory cytokines. The gene expression pattern of IL-1 is
similar to iNOS expression with comparable induction kinetics. Neither lipid
vesicles alone nor the combination with either AS or control ODN showed any
impact on the de novo mRNA expression of IL-1
during cytokine activation
(Fig. 5A). Therefore,
AS-ODN-mediated inhibition of iNOS must be regarded as specific, not
interfering with general cytokine inducibility.
Next, the degree of AS inhibition on protein expression was studied using Western blotting (Fig. 5C). We find an almost complete block of iNOS protein formation by ASODN (Fig. 5C, lane 1), whereas control ODN had no effect (lane 2). The degree of inhibition for iNOS protein synthesis was better than 95% compared with cytokine treatment only. Again, a significant increase in iNOS protein formation by empty vehicle addition was observed (lane 4). In resident cells, iNOS protein was completely absent (lane 5).
To also assess the effect of AS-ODN on NO formation, nitrite accumulation in culture supernatants was measured. Treatment with AS-ODN caused a significant and dose-dependent decrease in nitrite formation without any effects of the two different control ODN (Fig. 6A). Using empty vehicles, again a highly relevant and dose-dependent increase in nitrite accumulation was found (Fig. 6B). In conclusion, with the protocol used here, we find a significant AS-ODN-mediated inhibition of iNOS on all levels of enzyme synthesis and activity when using Lipofectin as uptake enhancer.
DISCUSSION
Our experiments demonstrate for the first time that the use of AS-ODN packed into lipid vesicles indeed offers a tool to achieve a specific and high degree of iNOS inhibition in EC in an inflammatory setting. This is promising for in vivo trials, but our setting at the same time shows general problems and limitations of this technique.
In agreement with an increasing number of publications
(9,
11), we see neither uptake, as
monitored by fluorescence microscopy, nor inhibition of nitrite formation in
the absence of suitable vehicles. However, Lipofectin in contrast to
LipofectAMINE displays negligible toxicity in nontransformed cells and thus
represents a good solution for intracellular delivery of ODN. Surprisingly, we
always observed that the addition of Lipofectin alone significantly enhances
iNOS mRNA and protein expression, as well as NO formation, in activated EC
only. Because LPS contamination could not be detected using the Limulus lysate
assay, this effect appears to be due to the lipid molecules themselves by
incorporation of the charged lipid moieties into the cellular membranes. Our
experiments further show that vesicles loaded with ODN do not cause this
increase in iNOS expression and NO production, irrespective of the ODN
sequence or specificity; therefore, proper loading protocols should solve this
problem. Nevertheless, it still has to be taken into consideration that in an
inflammatory setting, the application of lipid-encapsulated AS-ODN might turn
an ODN-mediated iNOS knockdown into an upregulation of NO production, probably
due to interactions with charged lipids and cytokine-mediated signaling.
However, the iNOS-enhancing property of the vehicles used here appears to
represent a restrictive activity that does not act on all inflammatory
responses, because the cytokine-driven IL-1 expression is not altered by
empty vehicles. We have no explanation for this observation, and there are no
published data that would help to understand this currently. However, because
lipid segregation, i.e., lipid rafting, is known to modulate a number of
immune responses, this might be a possible mechanism triggered by introducing
loaded lipids into the cell membrane.
The exact mechanism of AS-mediated inhibition is still under debate, but in our experimental setup, we found a distinct correlation between intranuclear accumulation of ODN and successful inhibition of enzyme activities because nitrite production was reduced only when ODN had entered the nucleus. In the continuous presence of FCS, FITC-labeled ODN accumulate around the nuclear envelope and in the cytoplasm but not within the nuclear compartment. Even when formation of ODN-lipid complexes was performed in FCS-free medium and FCS was present only during cell culture incubation, ODN were not localized in nuclei. In cell culture systems, it poses no problem for creating a FCS-free or -depleted environment. However, under in vivo conditions with the constant presence of serum, it is questionable whether ODN or other gene transfer applications using vehicle-mediated uptake will still show the same effectiveness observed in cell culture.
With LipofectAMINE, excellent staining of nuclei with hardly any ODN left in the cytoplasm was seen, but, surprisingly, no inhibition of nitrite formation was observed. LipofectAMINE-ODN interactions might be responsible for this result, prohibiting ODN from getting in close contact with their target structures. Concerning the mechanism of AS inhibition, our results underline the finding that AS-ODN exert their main effect not on the level of mRNA but rather on protein formation. Indeed, the decrease in mRNA synthesis as seen with Lipofectin was found to never exceed a level of 40% relative to sham-treated controls, whereas inhibition of protein synthesis was higher than 95%. This finding suggests a direct action mainly on specific protein synthesis. One explanation for the differences in iNOS mRNA vs. protein inhibition might be seen in the blocking of iNOS mRNA transport into the cytoplasm, which would also explain the necessity of nuclear accumulation of ODN for successful action. A similar observation has been made with using iNOS-specific AS inhibition in mouse mixed glial cell cultures (7).
When investigating the impact on enzyme activity as assessed by
accumulation of nitrite in the culture supernatants, the degree of inhibition
did not completely reflect the nearly complete inhibition of protein
formation, again an observation also noted with the mouse glial cultures
(7). This may be explained by
cytokine-mediated induction of proteins with accessory functions on enzyme
activity, for instance, increased arginine transport via cytokine-inducible
CAT-2 (5), but lack of feedback
inhibition might also be responsible for the few enzyme molecules formed to
work at maximal activity. Potentially, increased endothelial NOS3 activity
could also contribute. Together, our data demonstrate a complex effect of
AS-ODN, affecting every level of iNOS-derived NO synthesis, albeit at
different degrees. However, suppression of iNOS expression can be regarded as
a specific process because no effect was found on concomitant endothelial
IL-1 expression, another inflammation marker also synthesized de novo in
the presence of proinflammatory stimuli, following a similar time pattern, and
in part using identical activation pathways such as NF-
B.
In conclusion, we show here that AS-ODN targeted to iNOS represent a powerful tool for studying endothelial dysfunctions and inflammatory disorders. Especially when considering the possible protective activity that more recently has been attributed to high-output NO synthesis via iNOS activity, the specific inhibition achieved through AS-ODN-lipid complexes will allow us to define beneficial vs. detrimental effects mediated by this early inflammatory response.
DISCLOSURES
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 503 A3 and SFB 575 B2.
ACKNOWLEDGMENTS
We thank Waltraud Finkberg for determining endotoxin levels and Ulla Lammersen and Marija Lenzen for excellent technical assistance.
Address for reprint requests and other correspondence: V. Kolb-Bachofen,
Research Group Immunobiology, Heinrich-Heine-Univ. of Düsseldorf,
Gebäude 23.12, Postfach 10 10 07, D-40001 Düsseldorf, Germany
(E-mail:
bachofen{at}uni-duesseldorf.de).
FOOTNOTES
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.
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