1Endothelial Cell and Vascular Drug Targeting Group, Medical Biology Section, Department of Pathology and Laboratory Medicine, 2Department of Rheumatology, 3Department of Pharmacokinetics and Drug Delivery, 4Department of Therapeutic Gene Modulation, and 5Department of Epidemiology and Bioinformatics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Submitted 17 December 2004 ; accepted in final form 14 June 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
inflammatory gene expression; anti-inflammatory drugs; pharmacology; combination treatment
The proinflammatory cytokines TNF- and IL-1
, having a similar but not identical effect on gene expression, are often present simultaneously in chronic inflammatory diseases (29, 31). They exert a prominent effect on the expression of proinflammatory genes in endothelial cells. This effect takes place predominantly through activation of intracellular signaling pathways involving NF-
B and p38 MAPK (13, 39). The transcription factor NF-
B is present in the cytoplasm of unstimulated cells in an inactive form because of its association with the inhibitory protein inhibitor
B (I
B). On cytokine activation, degradation of I
B and subsequent nuclear translocation of active NF-
B takes place (18). The p38 MAPK activation pathway engages diverse upstream kinases responsible for p38 MAPK activation as well as downstream substrates (26). In endothelial cells both NF-
B and p38 MAPK are involved in the regulation of the expression of genes encoding E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2, among others (7, 11, 30). The regulation takes place at transcriptional and posttranscriptional levels (28, 32, 39).
Both activated NF-B and p38 MAPK have been shown to be present in rheumatoid arthritis and inflammatory bowel disease lesions and are therefore interesting targets for pharmacological intervention (17, 33, 37, 40). However, inhibition of NF-
B or p38 MAPK can have the serious drawback of undesired toxic effects on nondiseased cells (3, 38). Incorporation of such inhibitors in endothelial cell-specific drug targeting systems can theoretically overcome these undesired side effects (8). The antioxidant and metal-chelating compound pyrrolidine dithiocarbamate (PDTC) (21), the glucocorticoid dexamethasone (Dex) (43), the thioredoxin inhibitor methyl-(4R/S)-4-hydroxy-4-[((5S,8S)/(5R,8R))-8-methyl-1,3-dioxo-2-phenyl-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]-2-butynoate (MOL-294) (19), and the p38 MAPK inhibitor 4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl)-3-butyn-1-ol (RWJ-67657) (27) are potential candidates for incorporation in drug targeting constructs. However, limited data are available on quantitative comparison of the effects of these anti-inflammatory drugs on endothelial cell gene expression under proinflammatory conditions.
In the present study we investigated the effects of TNF-, IL-1
, and a combination of TNF-
and IL-1
on the kinetics and levels of expression of the proinflammatory genes E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and COX-2 by human umbilical vein endothelial cells (HUVEC). The importance of NF-
B signaling in TNF-
- and IL-1
-induced gene expression was investigated by overexpression of an I
B mutant inhibiting NF-
B signal transduction. Furthermore, we analyzed the effects of the above-mentioned drugs on the expression levels of the proinflammatory genes and their capacity to potentiate their inhibitory effects when added simultaneously. To quantitatively compare the effects of activators and drugs, real-time RT-PCR analysis of mRNA levels and, in separate experiments, ELISA and flow cytometric analyses of the proteins produced were performed.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of HUVEC.
Confluent HUVEC were activated with 1 and 10 ng/ml TNF- (Boehringer, Ingelheim, Germany) and 1 and 10 ng/ml IL-1
(R&D Systems, Minneapolis, MN), added separately or in combination, for 6 h (early gene expression) and 24 h (late gene expression). After incubation cells were microscopically analyzed with regard to their morphology and consistently were found to be adherent and viable. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Promega Benelux, Leiden, The Netherlands) assays were executed according to the manufacturer's protocol to corroborate the light microscopy analysis.
dnIB adenovirus.
Recombinant, replication-deficient adenovirus Ad5I
BAA, hereafter referred to as dnI
B, was a gift from Dr. C. Trautwein from the Medical School of Hannover, Germany. Adenovirus contained an I
B
sequence, in which serine at positions 32 and 36 was substituted by alanine and which was fused to an influenza A virus hemagglutinin (HA) tag. The expression was controlled by the cytomegalovirus promoter/enhancer (15). Virus was grown on HEK293 cells and purified from cell lysates by banding twice on CsCl gradients. Virus was desalted with a 10-kDa slide-A-lyzer (Pierce Chemical, Rockford, IL) in HEPES-sucrose buffer, pH 8.0 and stored at 80°C. Viral particles (vp) were determined by UV spectrophotometric analysis at 260 nm. Furthermore, a standard limiting dilution assay was performed to determine the vp-to-plaque-forming unit (pfu) ratio. As a control, adenovirus Ad5LacZ, containing the Escherichia coli
-galactosidase gene (12), was grown and purified as described above.
Virus infection protocol.
For the transduction of HUVEC with dnIB or the control virus Ad5LacZ, HUVEC were plated at 12,500 cells/cm2 in six-well tissue culture plates (Costar) and cultured overnight before actual transduction. The viral vectors, diluted in DMEM (GIBCO, Paisley, UK) without FCS, were added at 500 pfu/seeded cell (corresponding to 7.5 x 103 vp/cell) and incubated for 90 min at 37°C. The incubation medium was then replaced by endothelial culture medium. Cells were subsequently incubated for 24 h before activation to allow transgene expression.
Western blot analysis of dnIB expression in HUVEC.
After 24 h of culturing, cells were detached from the surface by trypsin-EDTA treatment, lysed in the cell culture lysis reagent (Promega, Madison, WI), and sonicated twice at 4°C for 5 s. After centrifugation for 10 min at 10,000 g and 4°C, cleared cell lysates were collected, and the protein content was determined with Bradford protein assay reagent (Bio-Rad Laboratories, Hercules, CA), with BSA as the standard. Samples were then mixed with reducing SDS sample buffer and boiled for 5 min, and 30 µg of protein was loaded on SDS-PAGE 10% acrylamide gel. After separation proteins were electrophoretically transferred on a nitrocellulose membrane (Bio-Rad Laboratories). Blots were blocked in blocking buffer containing 5% nonfat dry milk in PBS-0.1% Tween for 2 h. Next, blots were incubated for 1 h with rabbit anti-HA-probe antibody (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200 in blocking buffer) for dnI
B detection and rabbit anti-I
B
antibody (Santa Cruz Biotechnology; dilution 1:20 in blocking buffer) for both endogenous and dnI
B detection. Blots were washed with PBS-0.1% Tween and incubated for 1 h with horseradish peroxidase-conjugated swine anti-rabbit antibody (Dako, Glostrup, Denmark) diluted 1:2,000 in blocking buffer. After washing as described above, detection was performed with enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, IL) according to the manufacturer's protocol.
Incubation of HUVEC with drugs.
The following drugs were used: PDTC (Sigma, Zwijndrecht, The Netherlands), Dex (9-fluoro-16
-methyl-11
,17
,21-trihydroxy-1,4-pregnadiene-3,20-dione; Genfarma, Maarssen, The Netherlands), MOL-294 (kindly provided by Dr. M. Kahn from Pacific Northwest Research Institute, Seattle, WA), and RWJ-67657 (kindly provided by Johnson & Johnson Pharmaceutical R&D, Raritan, NJ). Stock solutions (10 mM) of PDTC, Dex, MOL-294, and RWJ-67657 were prepared in DMSO (Merck, Darmstadt, Germany). The stock solutions were diluted in endothelial culture medium to final concentrations as indicated in each experiment.
Anti-inflammatory drugs were added to confluent HUVEC 1 h before activation by TNF- or IL-1
. After 6 and 24 h of stimulation cells were analyzed microscopically with regard to morphology and viability, after which cells or supernatants were subjected to further analysis. The occurrence of toxic effects of drugs to the cells was excluded by MTS assay.
RNA isolation and real-time RT-PCR analysis. Total RNA was isolated with an Absolutely RNA Microprep Kit (Stratagene, Amsterdam, The Netherlands) according to the protocol of the manufacturer. RNA was analyzed qualitatively by gel electrophoresis and quantitatively with a RiboGreen RNA Quantitation Kit (Molecular Probes Europe, Leiden, The Netherlands). One microgram of total cellular RNA was subsequently used for the synthesis of first-strand cDNA with SuperScript III RNase H minus reverse transcriptase (Invitrogen, Breda, The Netherlands) in a 20-µl final volume containing 250 ng of random hexamers (Promega) and 40 units of RNase OUT inhibitor (Invitrogen). After RT reaction cDNA was diluted with distilled water to 100 µl, and 1 µl cDNA was used for each PCR reaction. Exons overlapping primers and minor groove binder (MGB) probes used for real-time RT-PCR were purchased as Assay-on-Demand from Applied Biosystems (Nieuwekerk a/d IJssel, The Netherlands): housekeeping gene GAPDH (assay ID Hs99999905_m1), endothelial cell marker CD31 (platelet endothelial cell adhesion molecule 1, Hs00169777_m1), E-selectin (Hs00174057_m1), VCAM-1 (Hs00365486_m1), ICAM-1 (Hs00164932_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), and COX-2 (Hs00153133_m1). The final concentration of primers and MGB probes in TaqMan PCR MasterMix (Applied Biosystems, Foster City, CA) for each gene was 900 and 250 nM, respectively. As controls, RNA samples not subjected to reverse transcriptase were analyzed to exclude unspecific signals arising from genomic DNA. Those samples consistently showed no amplification signals.
TaqMan real-time RT-PCR was performed in an ABI PRISM 7900HT Sequence Detector (Applied Biosystems). Amplification was performed with the following cycling conditions: 2 min at 50°C, 10 min at 95°C, and 4045 two-step cycles of 15 s at 95°C and 60 s at 60°C. Triplicate real-time RT-PCR analyses were executed for each sample, and the obtained threshold cycle values (Ct) were averaged. According to the comparative Ct method described in the ABI manual (http://www.appliedbiosystems.com), gene expression was normalized to the expression of the housekeeping gene GAPDH, yielding the Ct value. The average
Ct value obtained from resting, nontreated HUVEC was then subtracted from the average
Ct value of each sample subjected to the experimental conditions described, yielding the
Ct value. The gene expression level, normalized to the housekeeping gene and relative to the control sample, was calculated as 2
Ct.
IL-6 and IL-8 production measured by ELISA. In the designated drug combination treatment experiments, HUVEC medium was harvested, centrifuged, and stored at 20°C before cytokine quantification by ELISA. Ninety-six-well plates (Costar) were precoated with MoAb.CLB.MIL6/16 (CLB, Amsterdam, The Netherlands) diluted 1:1,000 in PBS for IL-6 and with MoAb.anti-IL-8 (R&D Systems) for IL-8 analysis. After blocking with 2% BSA-0.05% Tween in PBS, samples were incubated for 2 h in incubation buffer containing 0.2% gelatin-0.05% Tween in PBS. After being washed, bound IL-6 or IL-8 was detected with biotinylated polyclonal swine anti-human IL-6 (CLB) or polyclonal swine anti-human IL-8 (R&D Systems), respectively, in combination with streptavidin-E+ (CLB). Peroxidase activity was determined with tetramethylbenzidine (Roth, Karlsruhe, Germany) as substrate. IL-6 and IL-8 levels were calculated in the linear range of the assay from a standard curve (101,000 pg/ml) with recombinant human (rh)IL-6 (R&D Systems) and rhIL-8 (R&D Systems).
Flow cytometric analysis of cell adhesion molecule expression.
HUVEC pretreated with MOL-294 and/or RWJ-67657 as indicated were stimulated for 6, 12, and 24 h with TNF- and subsequently detached from the wells by a short treatment with trypsin. After being washed with PBS-5% FCS, cells were incubated for 45 min on ice with mouse monoclonal antibodies against human E-selectin (H18/7-acb), VCAM-1 (E1/6-aa2), and ICAM-1 (hu5/32.1) (all 3 antibodies kindly provided by Dr. M. A. Gimbrone from Harvard Medical School, Boston, MA) and CD31 (DakoCytomation, Glostrup, Denmark). After a PBS-5% FCS wash, detection was performed with FITC-conjugated rabbit anti-mouse antibody for 45 min on ice. Cells were fixed with 0.5% paraformaldehyde-PBS, after which flow cytometric analysis was performed on an Epics-Elite flow cytometer (Coulter Electronics, Mijdrecht, The Netherlands). A total of 5,000 events were analyzed per sample. Nonspecific staining was assessed by incubation with mouse IgG monoclonal antibodies specific for an irrelevant antigen as primary antibody. The mean fluorescence intensity values of these controls were consistently found to be <5.
Statistical analysis. Statistical significance of differences for experiments with adenovirus and single cytokine or drug treatment was studied by means of the two-sided Student's t-test, assuming equal variances. Differences were considered to be significant when P < 0.05.
Linear mixed models and ANOVA were used for data analysis of combination treatment experiments to address the significance of observed effects. Sidak and Tukey adjustment methods were used to adjust the confidence intervals and significance values to account for multiple comparisons. All analyses were performed with SPSS 12.01. Differences were considered to be significant when P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Between experiments, basal Ct values of proinflammatory genes in nonstimulated HUVEC samples ranged between 1 and 3 Ct units, whereas for each gene the Ct value obtained after 6 h of cytokine activation reached levels similar in each experiment (data not shown). These results imply that although the basal expression levels of genes might differ because of the heterogeneity of endothelial cells obtained from different donors, the activation status achieved by cytokine signaling was similar in all experiments. Expression of the endothelial cell marker CD31 remained constant under all conditions studied (Table 1), a result corroborating previously published studies (22). Patterns of proinflammatory gene expression, however, differed markedly with regard to the activator used and the incubation time studied (Fig. 1 and Table 1). At both time points studied TNF- induced the expression of adhesion molecules VCAM-1 and ICAM-1 to a higher extent than IL-1
, whereas IL-1
more profoundly affected IL-6, IL-8, and COX-2. At 6 h, the level of the gene expression was independent of the concentration of each activator, the only deviation being IL-1
-induced IL-6 and ICAM-1 expression (P > 0.05). Notably, for TNF-
-induced activation at 24 h, a higher concentration of TNF-
induced gene expression to a higher level (only for TNF-
-induced COX-2 and IL-6; P > 0.05). In time, E-selectin, VCAM-1, and ICAM-1 expression levels diminished, whereas IL-8 and COX-2 mRNA levels increased (P < 0.05). IL-6 exhibited a mixed response, as its level of expression induced by TNF-
was significantly higher at 24 h, whereas IL-1
-induced expression was significantly higher at 6 h.
|
|
|
|
Effects of chemical inhibitors of intracellular signaling pathways on inflammatory gene expression.
From the experiments performed with dnIB-expressing HUVEC, a possible involvement of (an)other cell activation pathway(s) in regulation of cytokine-induced inflammatory gene expression became apparent. Therefore, we investigated the effects of NF-
B inhibitors with different molecular targets and of one p38 MAPK inhibitor on proinflammatory gene expression. HUVEC were incubated with 1 µM of PDTC (data not shown), Dex, MOL-294, or RWJ-67657 1 h before the addition of 10 ng/ml TNF-
or IL-1
. The choice of this fixed concentration of drugs was based on the experience that 1 µM is a drug concentration that could be achieved with drug targeting constructs (9). The minor modulatory effects of PDTC on gene expression (data not shown) were possibly due to the use of a relatively low concentration of this drug compared with >10 µM concentrations used in other studies (21). As shown in Fig. 4, the drugs affected inflammatory gene expression differently depending on the activator used and the time interval studied. RWJ-67657 and MOL-294 were the most potent inhibitors, showing downregulation of several inflammatory genes. Inhibition of p38 MAPK activity by RWJ-67657 resulted in blocking of gene expression of the interleukins and COX-2 at 6 h and additionally of the adhesion molecules after 24 h. In contrast, MOL-294 treatment resulted in an inhibitory effect on both TNF-
- and IL-1
-induced adhesion molecule expression, with the most pronounced effect on VCAM-1 expression at both 6 and 24 h. We consistently found significantly higher IL-8 mRNA levels at 6 h after TNF-
treatment in combination with this drug. Dex downregulated COX-2 gene expression in all conditions studied for
2550%, TNF-
-induced IL-6 gene expression after 6 h of TNF-
activation, and VCAM-1 mRNA levels after 24 h of IL-1
activation. Moreover, pretreatment of HUVEC with 1 µM Dex resulted in a significant increase in IL-6 mRNA level 24 h after IL-1
stimulation compared with untreated cells.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that the pattern of proinflammatory gene expression markedly differed depending on the activator used. IL-1 mainly induced IL-6, IL-8, and COX-2 gene expression in HUVEC, an effect possibly due to more efficient utilization of p38 MAPK or other routes that control NF-
B-dependent expression (32, 39, 45). On the other hand, TNF-
more profoundly affected the expression of cell adhesion molecules. These data are in line with those reported by others (39, 45), although the latter studies were not performed in a quantitative manner and did not include a direct comparison of all genes as demonstrated in our study.
TNF- and IL-1
can be present simultaneously in proinflammatory diseases (29, 31). Because limited data are available on endothelial cell proinflammatory gene expression after simultaneous cytokine treatment (5, 6), we studied the effects of TNF-
+ IL-1
cotreatment on gene expression by HUVEC. As long as a combinatory activation does not saturate common cofactors, TNF-
and IL-1
partly utilize different signaling pathways (2). Therefore, one could expect that cytokine cotreatment would induce gene expression to a level that would be the sum of the levels obtained by either cytokine treatment alone. Interestingly, less than additive mRNA levels of adhesion molecules and additive or even synergistic levels of interleukins and COX-2 mRNA were observed. The less than additive effects on cell adhesion molecules might be explained by saturation of the molecules that control gene expression at low concentrations of either TNF-
or IL-1
(see also Fig. 1); other phenomena likely underlie the effects observed for the cytokines and COX-2. In dendritic cells it was shown that lipopolysaccharide was able to strongly activate p38 MAPK, an event instrumental for efficient phosphorylation and phosphoacetylation of histone H3. This marked IL-6 and IL-8 promoters for increased NF-
B recruitment, thereby enhancing the gene expression induction of these cytokines. Direct activation of p38 MAPK by TNF-
receptor signaling was weak, resulting in minor recruitment of p65 to IL-6 and IL-8 promoters (32). We showed that IL-1
-induced IL-6, IL-8, and COX-2 gene expression was stronger than their TNF-
-induced expression and that IL-6, IL-8, and COX-2 are under p38 MAPK control to a significant extent (Figs. 4 and 5). Possibly, also in endothelial cells IL-1
- and TNF-
-induced p38 MAPK and NF-
B collaborate in a more pronounced transcriptional induction of the cytokine genes. Conversely, ICAM-1 expression is almost completely p38 MAPK independent (Fig. 4 and Ref. 10); thus its expression is likely not affected by histone phosphorylation/acetylation-dependent NF-
B recruitment. The observation that on TNF-
+ IL-1
cotreatment ICAM-1 mRNA levels were less than additive fits this model of facilitated transcription. Because the molecular mechanisms of the observed attenuation/increases in gene expression induced by proinflammatory activator cotreatment are not completely clear at present, we decided to perform our subsequent pharmacological studies while using TNF-
and IL-1
separately. Still, this observation justifies further research, as it has important implications for the choice of the experimental conditions to study the pharmacological potency of new chemical entities in the drug development pipeline.
Both the dnIB adenovirus and drug treatment experiments confirmed a major role of the NF-
B and p38 MAPK pathways in regulation of gene expression in HUVEC as part of the inflammatory response induced by TNF-
and IL-1
(7, 11, 30, 39). An almost total inhibition of adhesion molecule expression in dnI
B-expressing HUVEC was observed at both early and late time points of activation. The induction of IL-6 and IL-8 expression was also inhibited by dnI
B, but to a lower extent than the effects on the adhesion molecules, indicating considerable involvement of (an)other pathway(s) regulating IL-6 and IL-8. Ridley et al. (30) reported that the expression of IL-6 and IL-8 induced by IL-1
in HUVEC was p38 MAPK dependent. In another study, TNF-
- and IL-1
-induced IL-8 gene and protein expression were shown to be partly dependent on reactive oxygen species generation and activator protein 1 activation (14, 44). Also, from our pharmacological experiments, the conclusion seems justified that p38 MAPK is, at least partly, controlling expression of these genes.
An interesting example of uncoupling of gene and protein expression was noted at the early time point in MOL-294-pretreated cells, where a consistent increase in IL-8 mRNA together with a significant block of IL-8 protein production was observed. Possibly, inhibition of thioredoxin resulted in an increased production of reactive oxygen species, thereby increasing IL-8 mRNA expression (14). The inhibition of IL-8 protein production induced by MOL-294, on the other hand, may be due to the fact that thioredoxin is required for efficient proteolysis catalyzed by thiol-dependent Cys proteases such as cathepsin (16). Cathepsins are known to be essential in processing of mature IL-8 protein at inflammatory sites (25). In Fig. 7 a schematic representation of the pathways studied and their possible interaction is given.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Chariot A, Meuwis MA, Bonif M, Leonardi A, Merville MP, Gielen J, Piette J, Siebenlist U, and Bours V. NF-B activating scaffold proteins as signaling molecules and putative therapeutic targets. Curr Med Chem 10: 593602, 2003.[CrossRef][ISI][Medline]
3. Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF, and Karin M. The two faces of IKK and NF-B inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion. Nat Med 9: 575581, 2003.[CrossRef][ISI][Medline]
4. Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, and Stern DM. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91: 35273561, 1998.
5. Dagia NM and Goetz DJ. A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1- and TNF-
-induced ECAM expression and adhesion. Am J Physiol Cell Physiol 285: C813C822, 2003.
6. Daxecker H, Raab M, Markovic S, Karimi A, Griesmacher A, and Mueller MM. Endothelial adhesion molecule expression in an in vitro model of inflammation. Clin Chim Acta 325: 171175, 2002.[CrossRef][ISI][Medline]
7. Denk A, Goebeler M, Schmid S, Berberich I, Ritz O, Lindemann D, Ludwig S, and Wirth T. Activation of NF-B via the I
B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells. J Biol Chem 276: 2845128458, 2001.
8. Everts M, Kok RJ, Ásgeirsdóttir SA, Melgert BN, Moolenaar TJ, Koning GA, van Luyn MJ, Meijer DK, and Molema G. Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. J Immunol 168: 883889, 2002.
9. Everts M, Koning GA, Kok RJ, Ásgeirsdóttir SA, Vestweber D, Meijer DK, Storm G, and Molema G. In vitro cellular handling and in vivo targeting of E-selectin-directed immunoconjugates and immunoliposomes used for drug delivery to inflamed endothelium. Pharm Res 20: 6472, 2003.[CrossRef][ISI][Medline]
10. Goebeler M, Kilian K, Gillitzer R, Kunz M, Yoshimura T, Brocker EB, Rapp UR, and Ludwig S. The MKK6/p38 stress kinase cascade is critical for tumor necrosis factor--induced expression of monocyte-chemoattractant protein-1 in endothelial cells. Blood 93: 857865, 1999.
11. Gustin JA, Pincheira R, Mayo LD, Ozes ON, Kessler KM, Baerwald MR, Korgaonkar CK, and Donner DB. Tumor necrosis factor activates CRE-binding protein through a p38 MAPK/MSK1 signaling pathway in endothelial cells. Am J Physiol Cell Physiol 286: C547C555, 2004.
12. Herz J and Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci USA 90: 28122816, 1993.
13. Hoefen RJ and Berk BC. The role of MAP kinases in endothelial activation. Vascul Pharmacol 38: 271273, 2002.[CrossRef][ISI][Medline]
14. Hwang YS, Jeong M, Park JS, Kim MH, Lee DB, Shin BA, Mukaida N, Ellis LM, Kim HR, Ahn BW, and Jung YD. Interleukin-1 stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells. Oncogene 23: 66036611, 2004.[CrossRef][ISI][Medline]
15. Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, and Brenner DA. NFB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 101: 802811, 1998.
16. Kerblat I, Drouet C, Chesne S, and Marche PN. Importance of thioredoxin in the proteolysis of an immunoglobulin G as antigen by lysosomal Cys-proteases. Immunology 97: 6268, 1999.[CrossRef][ISI][Medline]
17. Marok R, Winyard PG, Coumbe A, Kus ML, Gaffney K, Blades S, Mapp PI, Morris CJ, Blake DR, Kaltschmidt C, and Baeuerle PA. Activation of the transcription factor nuclear factor-B in human inflamed synovial tissue. Arthritis Rheum 39: 583591, 1996.[ISI][Medline]
18. May MJ and Ghosh S. Signal transduction through NF-B. Immunol Today 19: 8088, 1998.[CrossRef][ISI][Medline]
19. Misra-Press A, McMillan M, Cudaback E, Qabar M, Ruan F, Nguyen M, Vaisar T, Nakanishi H, and Kahn M. Identification of a novel inhibitor of the NF-B pathway. Curr Med Chem 1: 2939, 2003.
20. Mulder AB, Blom NR, Smit JW, Ruiters MH, van der Meer J, Halie MR, and Bom VJ. Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase. Thromb Res 80: 399411, 1995.[CrossRef][ISI][Medline]
21. Munoz C, Pascual-Salcedo D, Castellanos MC, Alfranca A, Aragones J, Vara A, Redondo MJ, and de Landazuri MO. Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-B and AP-1 transcription factors activity. Blood 88: 34823490, 1996.
22. Murakami S, Morioka T, Nakagawa Y, Suzuki Y, Arakawa M, and Oite T. Expression of adhesion molecules by cultured human glomerular endothelial cells in response to cytokines: comparison to human umbilical vein and dermal microvascular endothelial cells. Microvasc Res 62: 383391, 2001.[CrossRef][ISI][Medline]
23. Nielsen OH, Vainer B, Madsen SM, Seidelin JB, and Heegaard NH. Established and emerging biological activity markers of inflammatory bowel disease. Am J Gastroenterol 95: 359367, 2000.[CrossRef][ISI][Medline]
24. Ogawara K, Rots MG, Kok RJ, Moorlag HE, Van Loenen AM, Meijer DK, Haisma HJ, and Molema G. A novel strategy to modify adenovirus tropism and enhance transgene delivery to activated vascular endothelial cells in vitro and in vivo. Hum Gene Ther 15: 433443, 2004.[CrossRef][ISI][Medline]
25. Ohashi K, Naruto M, Nakaki T, and Sano E. Identification of interleukin-8 converting enzyme as cathepsin L. Biochim Biophys Acta 1649: 3039, 2003.[ISI][Medline]
26. Ono K and Han J. The p38 signal transduction pathway: activation and function. Cell Signal 12: 113, 2000.[CrossRef][ISI][Medline]
27. Parasrampuria DA, de Boer P, Desai-Krieger D, Chow AT, and Jones CR. Single-dose pharmacokinetics and pharmacodynamics of RWJ 67657, a specific p38 mitogen-activated protein kinase inhibitor: a first-in-human study. J Clin Pharmacol 43: 406413, 2003.
28. Pietersma A, Tilly BC, Gaestel M, de Jong N, Lee JC, Koster JF, and Sluiter W. p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level. Biochem Biophys Res Commun 230: 4448, 1997.[CrossRef][ISI][Medline]
29. Redlich K, Schett G, Steiner G, Hayer S, Wagner EF, and Smolen JS. Rheumatoid arthritis therapy after tumor necrosis factor and interleukin-1 blockade. Arthritis Rheum 48: 33083319, 2003.[CrossRef][ISI][Medline]
30. Ridley SH, Sarsfield SJ, Lee JC, Bigg HF, Cawston TE, Taylor DJ, DeWitt DL, and Saklatvala J. Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels. J Immunol 158: 31653173, 1997.[Abstract]
31. Rogler G and Andus T. Cytokines in inflammatory bowel disease. World J Surg 22: 382389, 1998.[CrossRef][ISI][Medline]
32. Saccani S, Pantano S, and Natoli G. p38-Dependent marking of inflammatory genes for increased NF-B recruitment. Nat Immun 3: 6975, 2002.[CrossRef][ISI]
33. Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, Zenz P, Redlich K, Xu Q, and Steiner G. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 43: 25012512, 2000.[CrossRef][ISI][Medline]
34. Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN and Runge MS. Hypoxia induces cyclooxygenase-2 via the NF-B p65 transcription factor in human vascular endothelial cells. J Biol Chem 272: 601608, 1997.
35. Schroer K, Zhu Y, Saunders MA, Deng WG, Xu XM, Meyer-Kirchrath J, and Wu KK. Obligatory role of cyclic adenosine monophosphate response element in cyclooxygenase-2 promoter induction and feedback regulation by inflammatory mediators. Circulation 105: 27602765, 2002.
36. Smolen JS and Steiner G. Therapeutic strategies for rheumatoid arthritis. Nat Rev Drug Discov 2: 473488, 2003.[CrossRef][ISI][Medline]
37. Thiele K, Bierhaus A, Autschbach F, Hofmann M, Stremmel W, Thiele H, Ziegler R, and Nawroth PP. Cell specific effects of glucocorticoid treatment on the NF-Bp65/I
B
system in patients with Crohn's disease. Gut 45: 693704, 1999.
38. Van den Blink B, Juffermans NP, ten Hove T, Schultz MJ, van Deventer SJ, van der Poll T, and Peppelenbosch MP. p38 mitogen-activated protein kinase inhibition increases cytokine release by macrophages in vitro and during infection in vivo. J Immunol 166: 582587, 2001.
39. Viemann D, Goebeler M, Schmid S, Klimmek K, Sorg C, Ludwig S, and Roth J. Transcriptional profiling of IKK2/NF-B- and p38 MAP kinase-dependent gene expression in TNF-
-stimulated primary human endothelial cells. Blood 103: 33653373, 2004.
40. Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, and Schreiber S. p38 Mitogen-activated protein kinase is activated and linked to TNF- signaling in inflammatory bowel disease. J Immunol 168: 53425351, 2002.
41. Westra J, Kudo JM, van Rijswijk MH, Molema G, and Limburg PC. Chemokine production and E-selectin expression in activated endothelial cells are inhibited by p38 MAPK (mitogen activated protein kinase) inhibitor RWJ 67657. Int Immunopharmacol 5: 12591269, 2005.[CrossRef][ISI][Medline]
42. Wu G, Mannam AP, Wu J, Kirbis S, Shie JL, Chen C, Laham RJ, Sellke FW, and Li J. Hypoxia induces myocyte-dependent COX-2 regulation in endothelial cells: role of VEGF. Am J Physiol Heart Circ Physiol 285: H2420H2429, 2003.
43. Xu X, Otsuki M, Saito H, Sumitani S, Yamamoto H, Asanuma N, Kouhara H, and Kasayama S. PPAR and GR differentially down-regulate the expression of nuclear factor-
B-responsive genes in vascular endothelial cells. Endocrinology 142: 33323339, 2001.
44. Yamagishi S, Inagaki Y, Nakamura K, and Imaizumi T. Azelnidipine, a newly developed long-acting calcium antagonist, inhibits tumor necrosis factor--induced interleukin-8 expression in endothelial cells through its anti-oxidative properties. J Cardiovasc Pharmacol 43: 724730, 2004.[CrossRef][ISI][Medline]
45. Zhao B, Stavchansky SA, Bowden RA, and Bowman PD. Effect of interleukin-1 and tumor necrosis factor-
on gene expression in human endothelial cells. Am J Physiol Cell Physiol 284: C1577C1583, 2003.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |