HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 286/5/H2010    most recent 00481.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (26) Citing Articles via Google Scholar Google Scholar Articles by Ospina, J. A. Articles by Duckles, S. P. Search for Related Content PubMed PubMed Citation Articles by Ospina, J. A. Articles by Duckles, S. P.

Estrogen suppresses IL-1-mediated induction of COX-2 pathway in rat cerebral blood vessels

Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697-4625

Submitted 27 May 2003 ; accepted in final form 10 December 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Interleukin (IL)-1 is a potent inducer of inflammatory prostaglandins, which are important mediators of vascular response to cerebral injury, whereas estrogen reduces brain injury in models of ischemic stroke. Thus we examined the effects of in vivo IL-1 exposure on cerebrovascular cyclooxygenase (COX)-2 expression and function in an animal model of chronic estrogen replacement. Estrogen-treated and nontreated ovariectomized female rats received IL-1 injections (10 µg/kg ip), and then cerebral vessels were isolated for biochemical and contractile measurements. In estrogen-deficient rats, IL-1 induced cerebrovascular COX-2 protein expression; a peak response occurred 3 h after injection. COX-2 was localized to arterial endothelium using confocal microscopy. IL-1 increased PGE₂ but not PGI₂ production and decreased vascular tone as measured in isolated cerebral arteries; the latter effect was partially reversed by treatment with the selective COX-2 inhibitor NS-398 (10 µmol/l). In contrast, in animals treated with estrogen, IL-1 had no significant effect on COX-2 protein levels, PGE₂ production, or vascular tone. Combined treatment with 17-estradiol and medroxyprogesterone acetate also prevented increases in PGE₂ production after IL-1 treatment, but treatment with 17-estradiol had no effect. IL-1 induction of COX-2 protein was prevented by treatment with the nuclear factor-B inhibitor caffeic acid phenethyl ester (20 mg/kg ip), and estrogen treatment reduced cerebrovascular nuclear factor-B activity. Estrogen thus has potent anti-inflammatory effects with respect to cerebral vascular responses to IL-1. These effects may have important implications for the incidence and severity of cerebrovascular disease.

17-estradiol; cyclooxygenase-2; prostaglandin E₂; nuclear factor-B; interleukin-1

Cerebrovascular inflammation is an early event in the pathogenesis of stroke and is believed to contribute to secondary brain injury by promoting leukocyte infiltration into the brain (16), blood-brain-barrier injury (10), hyperemia (23), vasogenic edema, and increased intracranial pressure (12). Central to the initiation of inflammation is the synthesis of cytokines that signal the induction of proinflammatory mediators primarily through activation of nuclear factor-B (NF-B), a family of transcription factors that are ubiquitously involved in the coordinated expression of inflammation-related genes (28). In the central nervous system, interleukin (IL)-1 appears to be the predominant cytokine (12) and is released from multiple cell types, including astrocytes, neurons, microglia, and endothelial cells (39). It has been suggested (40) that IL-1 is an important contributor to the pathogenesis of stroke, because inhibition of IL-1 has been shown to reduce neurodegeneration after ischemic injury. However, a thorough understanding of the influence of IL-1 on cerebral circulation is lacking.

Cyclooxygenase-2 (COX-2) is an important mediator of vascular response to injury, and this enzyme is induced in response to cytokines, lipopolysaccharide (LPS), and ischemia-reperfusion (7). Once induced, COX-2 catalyzes the conversion of arachidonic acid to inflammatory prostaglandins, predominantly PGE₂ and PGI₂ (11). Vascular-derived prostanoids may be important mediators of cerebral vascular inflammation in that cyclooxygenase products promote cell adhesion molecule expression (46), postischemic hyperemia (31), blood-brain-barrier leakiness (51), and cytotoxicity (43). Conversely, estrogen can suppress leukocyte accumulation (41) and attenuate hyperemia (23) in the brain after an inflammatory insult. It was recently shown (17) that estrogen can suppress NF-B-dependent inflammation in cultured cerebral endothelial cells; however, a similar effect of estrogen in vivo has yet to be demonstrated. Taken together, these observations raise the possibility that estrogen suppresses cerebrovascular inflammation by regulating proinflammatory prostaglandin synthesis and function. Consequently, estrogen may confer benefit against ischemia-reperfusion brain injury by minimizing functional dysregulation of the cerebral circulation.

Therefore, in this study we examined the effects of in vivo IL-1 administration on COX-2 pathway expression, localization, and function in cerebral blood vessels of estrogen-deficient and -treated female rats. The latter group, ovariectomized females chronically treated with 17-estradiol, also was compared with animals treated with either the inactive enantiomer 17-estradiol or the clinically used combination of 17-estradiol and medroxyprogesterone acetate (MPA). In addition, we explored the role of NF-B in mediating induction of cerebrovascular COX-2 by IL-1 and whether its activation is influenced by in vivo 17-estradiol treatment. To our knowledge, this is the first study to provide evidence that 1) estrogen regulates cytokine-mediated induction of cerebrovascular inflammatory prostaglandin pathways in vivo, 2) NF-B participates in the induction of cerebrovascular COX-2 after peripheral IL-1 administration, and 3) chronic 17-estradiol treatment regulates vascular NF-B activation. These results suggest that in vivo estrogen treatment, by targeting NF-B activation, suppresses the inflammatory drive created by COX-2-derived products after exposure to inflammatory stimuli.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In vivo hormone treatment. All protocols involving the use of animals were approved by the Institutional Animal Care and Use Committee at the University of California, Irvine. Four groups of female rats were used in this study: ovariectomized (OVX), OVX treated with 17-estradiol (OVX+E), OVX treated with 17-estradiol (OVX+E), and OVX+E treated with MPA (OVX+E+MPA). Three-month-old female Fischer 344 rats (Charles River-SASCO Laboratories; Wilmington, MA) were ovariectomized while under anesthesia (46 mg/kg ip ketamine and 4.6 mg/kg ip xylazine). At the time of ovariectomy, some rats were subcutaneously implanted with Silastic capsules (inner diameter, 1.57; outer diameter, 3.18 mm) that contained either 17-estradiol (length, 5 mm), 17-estradiol (5 mm), or 17-estradiol (5 mm) and MPA (3 mm). Animals were allowed to recover from surgery and then were returned to a vivarium where they were housed in individual cages with free access to food and water in a temperature-controlled (22° C) room on a 12:12-h light-dark cycle.

Approximately 1 mo after surgery and hormone capsule implantation, animals were injected with IL-1 (10 µg/kg ip) or an equivalent volume of vehicle (0.9% saline ip). In some cases, OVX animals also received injections of 20 mg/kg ip caffeic acid phenethyl ester (CAPE; BIOMOL; Plymouth Meeting, PA) or vehicle [polyethylene glycol (PEG)-400] at 24, 12, and 1 h before IL-1 or saline treatment. At various time points (20, 60, 180, or 360 min) after IL-1 or saline injection, animals were anesthetized by CO₂, and blood samples were obtained by cardiac puncture for measurement of serum 17-estradiol by radioimmunoassay (Diagnostic Products; Los Angeles, CA). Alternatively, serum IL-1 levels were determined by ELISA (Assay Designs; Ann Arbor, MI). Although MPA levels were not quantitated, our lab has previously demonstrated that implantation of 3-mm-long MPA capsules in female rats results in MPA serum levels that are similar to those observed clinically (30). Rats then were killed by decapitation and their brains were quickly removed, immediately frozen in dry ice, and kept at –80° C until use. Alternatively, brains were used immediately to isolate blood vessels for prostanoid assay or tissue-bath experiments. The action of 17-estradiol was verified by measurement of whole body and dry uterine weights (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effects of treatment on 17-estradiol serum concentration, body weight, and uterine weight

Western blot analysis. COX-2 protein expression was examined in cerebral vessels from OVX and OVX+E rats 1, 3, or 6 h after peripheral IL-1 treatment or 3 h after saline injection. In addition, COX-2 protein was examined in CAPE- or PEG-400-treated animals 3 h after IL-1 exposure. To obtain cerebral vessels for Western blot analysis, brains were Dounce homogenized in ice-cold PBS (0.01 mol/l, pH 7.4) and centrifuged at 2,900 g for 5 min at 4° C. The supernatant was discarded and the pellet was washed several times by resuspension in PBS and subsequent centrifugation at 2,900 g for 5 min. The pellet was resuspended in PBS, layered over a 15% dextran (35–40 kDa; Sigma; St. Louis, MO) density-gradient solution, and finally centrifuged in a swinging-bucket rotor at 4,500 g for 25 min at 4° C. The aqueous supernatant was discarded, and the pellet that contained blood vessels was collected over a 50-µm nylon mesh and washed for several minutes with cold PBS. The isolated vessels, which were inspected by light microscopy, were a mixture of arteries, arterioles, veins, venules, capillaries, and associated perivascular elements (38).

Blood vessels were glass homogenized in lysis buffer (that contained 50 mmol/l -glycerophosphate, 100 µmol/l NaVO₃, 2 mmol/l MgCl₂, 1 mmol/l EGTA, 0.5% Triton X-100, 1 mmol/l DL-DTT, 20 µmol/l pepstatin, 20 µmol/l leupeptin, 0.1 U/ml aprotinin, and 1 mmol/l PMSF) and incubated on ice for 25 min. After homogenization, samples were centrifuged at 4,500 g for 5 min at 4° C. Supernatants that contained whole cell lysates were collected, and protein contents were determined via bicinchoninic acid (BCA) assay (Pierce).

Whole cell lysate protein samples (20 µg/lane) were loaded onto 8% Tris-glycine gels and separated via SDS-PAGE. Additionally, PMA/LPS-induced RAW 264.7 whole cell lysate (Santa Cruz Biotechnology; Santa Cruz, CA) and broad-range biotinylated molecular weight markers (Bio-Rad) were loaded for identification of protein bands. After electrophoretic separation, protein was transferred to nitrocellulose membranes (Amersham; Piscataway, NJ) in blocking buffer that contained 0.01 mol/l PBS with 0.1% Tween 20 (T-PBS) and 6.5% nonfat dry milk. Membranes were then incubated with the primary antibody of interest: rabbit polyclonal anti-COX-2 (1:600 dilution; Cayman Chemical; Ann Arbor, MI) or mouse monoclonal anti--actin (1:1,000 dilution; Sigma), followed by the appropriate secondary antibody: anti-rabbit IgG-horseradish peroxidase (1:7,500 dilution) or anti-mouse IgG-horseradish peroxidase (1:15,000 dilution; Transduction Laboratories). Bands were detected using electrochemiluminescence reagent (Amersham) and Hyperfilm (Amersham) and were quantitated with the computer-based electrophoresis analysis program UN-SCAN-IT (Silk Scientific; Orem, UT). Band densities were normalized to OVX (1 h) when the time course of COX-2 expression after IL-1 injection was examined or to OVX saline when the effects of IL-1 and saline on COX-2 levels were compared.

Immunohistochemical analysis. Scanning laser confocal microscopy was used to establish the cellular localization of COX-2 in cerebral arteries from OVX rats treated with IL-1 for 3 h. Immunohistochemical staining of cerebral arteries was performed as previously described (48). Pial arteries were dissected from the surface of the brain, cut into short segments, and fixed in 3% formaldehyde for 30 min. Arteries were then permeabilized with Triton X-100 (0.1%) for 5 min, placed in 1% bovine serum albumin (BSA)-PBS for 30 min, and coincubated with rabbit anti-COX-2 (1:50 dilution; Cayman Chemical) and mouse anti-endothelial nitric oxide synthase (eNOS, 1:50 dilution; Transduction Laboratories) antibodies in 0.1% BSA-PBS overnight at 4° C. Vessels then received three 10-min washes in PBS and were coincubated with secondary antibodies (10 µg/ml goat anti-rabbit Oregon Green-488 and goat anti-mouse Texas Red; Molecular Probes) overnight at 4° C. Arteries received three 10-min washes in PBS and were then placed on microscope slides, covered with VectaShield mounting medium that contained 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories) to counterstain nuclei, and coverslipped. Images were obtained using a Bio-Rad model 1024 laser scanning confocal microscope equipped with standard and UV lasers.

Prostanoid measurement. Prostanoid release from cerebral vessels of OVX, OVX+E, OVX+E, and OVX+E+MPA animals was measured after a 3-h in vivo treatment with IL-1 or saline. Freshly isolated brains were gently homogenized and washed in ice-cold 0.01 mol/l PBS (see Western blot analysis). Brain homogenates were passed over a 150-µm nylon mesh to separate the blood vessels (retained) from the rest of the brain tissue (washed through). After extensive washing, the vessels were collected and placed into HEPES-buffered saline (HBS, pH 7.4) that contained (in mmol/l) 122 NaCl, 1.6 CaCl₂, 15 HEPES, 5.2 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, and 11.5 dextrose (all from Fisher Scientific; Pittsburgh, PA) and equilibrated in a tissue-culture incubator (at 37° C in 95% O₂-5% CO₂) for 1 h. After the equilibration period, tissues were placed into fresh HBS at 37° C (in 95% O₂-5% CO₂) and incubated for 30 min. Separate aliquots of cerebral vessels from each brain were incubated in the presence and absence of 10 µmol/l NS-398 (Cayman Chemical) to assess the specific contribution of COX-2 to prostanoid production. After incubation, the medium was collected and immediately stored frozen at –20° C until prostanoid measurement. Tissues were lysed and protein contents were determined via BCA assay. Enzyme immunoassay was used to measure PGE₂ directly (Assay Designs) or to measure PGI₂ as its stable metabolite 6-keto-PGF₁ (Amersham) according to the protocols provided by the manufacturers. PGE₂ and 6-keto-PGF₁ values were normalized to tissue protein concentrations. COX-2-derived PGE₂ production quantities were determined as the difference in PGE₂ levels obtained from tissues incubated in the absence and presence of 10 µmol/l NS-398.

Contractile studies of pressurized arteries. Vascular tone was measured in cerebral arteries from OVX and OVX+E rats killed 3 h after injection of IL-1 or saline. Freshly isolated brains were immediately placed in ice-cold physiological saline solution (PSS) that contained (in mmol/l) 122 NaCl, 1.6 CaCl₂, 25.6 NaHCO₃, 5.2 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.03 EDTA, and 11.5 dextrose and were bubbled with 95% O₂-5% CO₂. Using a dissecting microscope, we isolated a 2- to 3-mm segment of a small-caliber branch of the middle cerebral artery from the area overlying the parietal cortex. Arteries with a passive intraluminal diameter of 130–140 µm at a pressure of 80 mmHg were chosen for this study. Once isolated, vessels were cleaned of any adhering tissue and placed into an arteriograph that contained PSS (Living Systems Instrumentation; Burlington, VT). One end of the artery was mounted and secured onto a glass microcannula connected to a pressure-servo device and briefly perfused with PSS to remove any blood remaining in the vessel lumen. The distal end of the artery was mounted and secured onto a microcannula connected to a closed luer lock to maintain no-flow conditions. Arteries were subsequently pressurized to 60 mmHg and allowed to equilibrate for 1 h while being superfused with PSS maintained at 37° C and pH 7.4 and bubbled with 95% O₂-5% CO₂. Vessels that did not develop spontaneous tone during the initial equilibration period or that had a passive diameter >150 µm at 80 mmHg were discarded. Intraluminal diameter and transmural pressure values were monitored by computer-based analysis using MacLab and Chart 3.6 software.

After equilibration, the autoregulatory response in cerebral arteries was assessed. Transmural pressure was decreased to 20 mmHg and diameter was allowed to stabilize over a 5- to 10-min period. Pressure was then gradually increased to 80 mmHg and the vessel was allowed to reach a stable diameter. Pressure was then returned to 60 mmHg and arteries were superfused with PSS that contained the selective COX-2 inhibitor NS-398 (10 µmol/l; Ref. 37) for 30 min. Pressure was again decreased to 20 mmHg and then gradually increased to 80 mmHg, and vessel diameters were recorded. At the conclusion of all experiments, vessels were superfused for 5–10 min with Ca²⁺-free PSS that contained 3 mmol/l EDTA, and the passive intraluminal diameters were measured at 20 and 80 mmHg. Vascular tone was defined as the difference between the passive (EDTA treated) and active (PSS or NS-398 treated) diameters. Autoregulatory response was determined as the difference in vascular tone between 80 and 20 mmHg.

NF-B p65 measurement. NF-B p65 DNA-binding activity was measured in nuclear extracts of cerebral vessels from OVX and OVX+E rats killed 20 min after injection of IL-1 or saline. Nuclear and cytosolic extracts were prepared as described previously (20). Briefly, cerebral blood vessels were isolated, finely diced on a glass slide that was kept over dry ice, Dounce homogenized in ice-cold buffer A (that contained 10 mmol/l HEPES, 1.5 mmol/l MgCl₂, 10 mmol/l KCl, 1 mmol/l DL-DTT, 1 mmol/l PMSF, and 0.1% Nonidet P-40, pH 7.9), and incubated on ice for 15 min. After homogenization, samples were centrifuged at 870 g for 10 min at 4° C. Pellets were resuspended in buffer A without Nonidet P-40 and recentrifuged at 870 g for 10 min at 4° C. Supernatants were discarded, and pellets were suspended in TransAm lysis buffer (Active Motif; Carlsbad, CA) and shaken for 30 min at 4° C. Suspensions were then microcentrifuged at 14,000 g for 10 min at 4° C. Supernatants that contained nuclear extracts were collected, and protein contents were determined via BCA assay.

NF-B p65 DNA binding was measured in nuclear extracts with a TransAM NF-B p65 immunoassay-based kit (Active Motif). Nuclear protein or HeLa whole cell extracts for positive control (2.5 µg/well) were incubated for 1 h in 96-well plates to which a double-stranded NF-B consensus oligonucleotide sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3' and 3'-TCAACTCCCCTGAAAGGGTCCG-5'; binding site underlined) had been conjugated (Active Motif). Activated NF-B p65 was detected by 1-h incubation with an anti-p65 antibody that recognizes an epitope as accessible only when NF-B is bound to DNA. This was followed by 1-h incubation with a horseradish peroxidase-conjugated secondary antibody and finally by exposure to a 3,3',5,5'-tetramethylbenzidine substrate solution. Reactions were measured by colorimetric methods, and data were expressed as the percent of the positive control signal.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with GraphPad Prism 2.0 software. Differences were assessed by one-way ANOVA or ANOVA with repeated measures where appropriate. Pairwise comparisons were made using Newman-Keuls post hoc analysis. Alternatively, Student's t-test was used where appropriate. For all comparisons, statistical significance was set at P 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In vivo model of hormone treatment. We used an in vivo rat model to explore the effects of chronic estrogen deficiency (OVX) or chronic treatment with 17-estradiol (OVX+E), 17-estradiol (OVX+E), or 17-estradiol plus MPA (OVX+E+MPA) on the induction of cerebrovascular COX-2 pathways after peripheral IL-1 treatment. To verify our model, serum 17-estradiol levels, whole body weights, and dry uterine weights were measured 1 mo after surgical treatment (Table 1). Serum from OVX+E or OVX+ E+MPA rats displayed levels of 17-estradiol that approximated those found in normally cycling rats (29); these levels were significantly greater than in OVX animals. Animals treated with 17-estradiol had serum 17-estradiol levels similar to those detected in OVX rats. In addition, OVX+E and OVX+E+MPA rats had significantly lower whole body weights and greater uterine weights compared with OVX rats, although MPA attenuated the stimulatory effect of estrogen on uterine growth. Treatment with 17-estradiol resulted in a small decrease in body weight and a slight increase in uterine weight; both measures were significantly different than those found in 17-estradiol-treated rats. These results suggest that implanted pellets sustained the physiological levels of 17-estradiol in the serum throughout the treatment period. Furthermore, circulating 17-estradiol was biologically active, and its trophic effects on the uterus were partially antagonized by MPA.

IL-1 induction of cerebrovascular COX-2. We first determined the effects of circulating IL-1 on the cerebrovascular COX-2 pathway in OVX+E and OVX rodents. We initially examined the time course of COX-2 protein expression in cerebral vessels for the 6-h period after peripheral administration of IL-1. Western blots for cerebral vessels from OVX and OVX+E animals treated with IL-1 were probed with anti-COX-2 polyclonal antibodies, and bands migrating at 72 kDa were detected (Fig. 1). These bands corresponded to the expected molecular weight of COX-2 and comigrated with the positive control used. COX-2 protein was detectable 1 h after IL-1 treatment; however, at this time point, the levels of COX-2 were not different between OVX and OVX+E animals. At 3 and 6 h after IL-1 exposure, the levels of COX-2 protein were significantly increased in vessels from OVX animals (Fig. 1). In contrast, in OVX+E animals, levels of cerebrovascular COX-2 remained unchanged at 3 and 6 h after cytokine injection (Fig. 1). COX-2 levels were thus significantly different between OVX and OVX+E rats at both 3 and 6 h after IL-1 treatment.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Effects of chronic 17-estradiol treatment on the time course of cerebrovascular cyclooxygenase-2 (COX-2) protein expression at 1, 3, and 6 h after peripheral interleukin (IL)-1 injection. A: a representative Western blot illustrates differences in COX-2 immunoreactivity in cerebral vessel lysates from ovariectomized (OVX) rats and OVX rats treated with 17-estradiol (OVX+E) after IL-1 administration in vivo. Bands migrating at 72 and 42 kDa were detected using antibodies directed against COX-2 and -actin, respectively. B: COX-2 densitometric data are illustrated as fold changes vs. values for OVX at 1 h. Values represent means ± SE; n = 3 rats; *P 0.05, significantly different from OVX animals at 1 h; #P 0.05, significantly different from OVX+E rats at equivalent time points.

COX-2 localization in cerebral arteries after IL-1 treatment. Laser scanning confocal microscopy was used to determine the cellular localization of COX-2 in cerebral arteries isolated from OVX rats 3 h after IL-1 injection. Using the orientation of DAPI-stained nuclei as a guide (2), we identified the smooth muscle cell layer by nuclei oriented perpendicular to the direction of blood flow (Fig. 2A) and the endothelial cell layer by nuclei oriented in the direction of blood flow (Fig. 2B). Once the vascular cell layer was identified, immunofluorescent staining for COX-2 (green) and the endothelial marker eNOS (red) was examined in the same focal plane. The eNOS was visualized in the endothelium (Fig. 2D) but not the smooth muscle cells (Fig. 2C) of cerebral arteries. Similarly, COX-2 immunoreactivity was revealed in cerebral artery endothelial cells (Fig. 2F) but not in smooth muscle cells (Fig. 2E). Merging all three images at the endothelial level (Fig. 2H) illustrates a partial colocalization of eNOS and COX-2 adjacent to the nuclei. In contrast, merging of images taken at the level of cerebral artery smooth muscle (Fig. 2E) demonstrates no localization of eNOS or COX-2. We previously found no detectable fluorescence when the secondary antibodies were used alone (C. Stirone, S. P. Duckles, and D. N. Krause, unpublished observation).

View larger version (90K): [in this window] [in a new window]   Fig. 2. Laser scanning confocal microscopic localization of COX-2 in a cerebral artery from an OVX rat 3 h after treatment with IL-1. A, C, and E: a single focal plane through the smooth muscle cell layer was imaged for either nuclei (blue), endothelial nitric oxide synthase (eNOS, red), or COX-2 (green), respectively. G: merged image of A, C, and E demonstrates no eNOS or COX-2 staining at the level of arterial smooth muscle. B, D, and F: a different focal plane through the endothelial cell layer of the same artery illustrates nuclear, eNOS, and COX-2 staining, respectively. H: merged image of B, D, and F shows colocalization of eNOS and COX-2 in the endothelium of cerebral arteries.

Estrogen regulation of IL-1 induction of COX-2. We next compared the effects of vehicle (0.9% saline) and IL-1 on cerebrovascular COX-2 induction 3 h after injection, as at this time the effects of IL-1 were most salient. As shown in Fig. 3, COX-2 band densities were expressed at very low yet similar levels after treatment of OVX or OVX+E animals with saline. Administration of IL-1, however, significantly increased COX-2 levels in OVX rats. In contrast, there was no cytokine effect in OVX+E animals. As expected, -actin levels were similar between all treatment groups.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Effects of 17-estradiol treatment on cerebrovascular COX-2 protein levels 3 h after IL-1 or saline control injection. A: a representative Western blot illustrates differences in COX-2 immunoreactivity in cerebral vessel lysates from OVX and OVX+E animals 3 h after IL-1 or saline administration. Bands migrating at 72 and 42 kDa were detected using antibodies directed against COX-2 and -actin, respectively. B: COX-2 densitometric data are illustrated as fold changes vs. OVX saline values. Values are means ± SE; n = 3 rats; *P 0.05, significantly different.

Effects of estrogen on serum IL-1. Estrogen does not appear to influence the in vivo degradation of IL-1 in that there were no significant differences in serum IL-1 concentrations between OVX (3.8 ± 0.9 ng/ml; n = 3) and OVX+E (2.9 ± 1.9 ng/ml; n = 4) animals 20 min after IL-1 injection. Effects of estrogen, MPA, and IL-1 on cerebrovascular PGE₂ production. We next assessed whether treatment of rats with IL-1 stimulated the elaboration of inflammatory prostaglandins, primarily PGE₂, from cerebral vessels incubated in vitro, and if so, whether prostanoid levels were influenced by prior chronic in vivo treatment with 17-estradiol, 17-estradiol, or 17-estradiol and MPA. Consistent with our previous study, there were no significant differences in PGE₂ levels among the saline-treated groups (38). Figure 4 illustrates that prior in vivo IL-1 treatment significantly increased in vitro cerebrovascular PGE₂ release from vessels of OVX animals compared with vessels from saline-treated controls. Chronic in vivo 17-estradiol treatment, however, prevented the inductive effects of IL-1 on cerebrovascular COX-2-derived PGE₂, and this preventive effect of 17-estradiol was preserved in vessels from animals simultaneously treated with MPA. On the other hand, when ovariectomized rats were chronically treated with 17-estradiol (which is the inactive enantiomer of 17-estradiol), IL-1 significantly stimulated cerebrovascular COX-2-derived PGE₂ production to levels comparable to those seen in vessels from OVX animals. Consequently, in vitro cerebrovascular PGE₂ production 3 h after in vivo IL-1 exposure was significantly greater in vessels from OVX and OVX+E but not OVX+E+MPA animals compared with OVX+E rats (Fig. 4).

View larger version (36K): [in this window] [in a new window]   Fig. 4. Effects of 17-estradiol (OVX+E), 17-estradiol (OVX+E), or 17-estradiol and medroxyprogesterone acetate (OVX+E+MPA) treatment on cerebrovascular production of PGE2 after 3 h in vivo treatment with IL-1. Cerebral vessels released PGE2 into the incubation medium, which was collected after 30 min. PGE2 was measured by enzyme immunoassay and normalized to the amount of protein in tissue samples. COX-2-derived PGE2 production was determined as the difference in PGE2 levels obtained from tissue incubated in the absence and presence of 10 µmol/l NS-398. Values are means ± SE; n = 4–6 rats; *P 0.05, significantly different from saline in same animal group; #P 0.05, significantly different from OVX+E IL-1.

Because COX-2 is believed to be a major source of prostacyclin in vascular cells, we investigated the possibility that IL-1 upregulates prostacyclin release. However, IL-1 treatment of OVX animals did not increase cerebrovascular prostacyclin production. Rather, there was a trend toward decreased release of PGI₂ in vessels from IL-1-treated animals (518 ± 120 pg/mg protein; n = 3) compared with saline-treated controls (910 ± 127 pg/mg protein; n = 3). These differences, however, did not reach statistical significance.

Effects of estrogen and IL-1 on tone of cerebral arteries. Next we determined the effects of peripheral IL-1 or saline administration to OVX and OVX+E animals on tone and autoregulation of pressurized small-caliber cerebral arteries isolated from these animals. As shown in Fig. 5A, 3 h after IL-1 administration to OVX rats, vascular tone in isolated cerebral arteries pressurized to 80 mmHg was significantly reduced compared with arteries isolated from saline-treated OVX rats. In contrast, in arteries from OVX+E rats, there were no differences between saline or IL-1 treatment with respect to cerebrovascular tone at 80 mmHg. However, cerebral vascular tone in both OVX+E groups was significantly greater than the OVX IL-1-treated group. Furthermore, in support of our previous work (37), arteries from OVX+E rats (saline treated) displayed significantly less tone than those from OVX saline-treated animals. At 80 mmHg, passive cerebral artery diameter measurements were nearly identical between saline- and IL-1-treated OVX and OVX+E animals (Fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effects of chronic estrogen treatment on vascular tone, passive diameter, and autoregulatory response of small-caliber cerebral arteries from animals treated with IL-1 or saline. A: vascular tone at 80 mmHg intraluminal pressure in cerebral arteries isolated from OVX and OVX+E rats 3 h after peripheral injection of IL-1 or saline. Tone was calculated as the difference between passive [determined in 3 mmol/l EDTA-Ca2+-free physiological saline solution (PSS)] and active (determined in PSS) diameters. B: passive intraluminal diameters determined in 3 mmol/l EDTA-Ca2+-free PSS. C: autoregulatory response was defined as the change in vascular tone between the application of 20 and 80 mmHg transmural pressure. In each case, vascular tone was calculated as the difference between passive (determined in 3 mmol/l EDTA-Ca2+-free PSS) and active (determined in PSS) diameters. Values are means ± SE; n = 4–7 rats; *P 0.05, significantly different from OVX saline treatment; #P 0.05, significantly different from OVX IL-1 treatment.

We also examined the changes in tone across a physiological range of intraluminal pressures. Figure 5C illustrates that increasing pressure from 20 to 80 mmHg resulted in increased tone that was not significantly different in magnitude between arteries from saline-treated OVX and OVX+E rats. In addition, there were no significant effects of IL-1 treatment on the autoregulatory response of cerebral arteries from either OVX or OVX+E animals (Fig. 5C).

Role of COX-2 in IL-1 effects on cerebrovascular tone. The contributions of COX-2 activity to IL-1-mediated changes in vascular tone were evaluated in pressurized cerebral arteries from OVX and OVX+E animals. As shown in Fig. 6A, in cerebral arteries from OVX animals treated with IL-1, selective pharmacological inhibition of COX-2 with NS-398 significantly increased tone and thus partially reversed the decrease in tone consequent to IL-1 treatment. Surprisingly, NS-398 caused a slight yet significant decrease in tone in cerebral arteries from OVX animals treated with saline (Fig. 6A). In contrast, cerebral arteries from OVX+E animals that received saline or IL-1 were unaffected by NS-398 (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effects of selective COX-2 inhibition with 10 µmol/l NS-398 on IL-1-mediated changes in vascular tone. A: vascular tone at 80 mmHg intraluminal pressure in cerebral arteries isolated from OVX rats 3 h after peripheral injection of IL-1 or saline to the animal. Tone was measured in PSS and after a 30-min suffusion of cerebral arteries with 10 µmol/l NS-398. Vascular tone was calculated as the difference between passive (determined in 3 mmol/l EDTA-Ca2+-free PSS) and active (determined in PSS) diameters. B: same as A but arteries were from OVX+E rats treated with IL-1 or saline. Values represent means ± SE; n = 4 or 5 rats; *P 0.05, significantly different from saline PSS; #P 0.05, significantly different from IL-1 PSS.

Role of NF-B in IL-1 induction of cerebrovascular COX-2: regulation by estrogen. We next explored whether NF-B signaling was involved in induction of cerebrovascular inflammatory prostanoid pathways after peripheral IL-1 administration. Figure 7 depicts the effects of in vivo pretreatment of OVX rats with the selective NF-B inhibitor CAPE (26, 33) before IL-1 injection. OVX animals treated with PEG-400 (the vehicle for CAPE) during the 24-h period preceding IL-1 administration expressed COX-2 in the cerebral vasculature at levels similar to those reported above (Fig. 7). CAPE (20 mg/kg) treatment for 24 h, however, abrogated the inductive effect of IL-1 on COX-2 and resulted in significantly lower levels of COX-2 protein expression.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Effects of nuclear factor (NF)-B inhibition on IL-1-mediated COX-2 induction in cerebral vessels from OVX rats. A: a representative Western blot of COX-2 immunoreactivity in cerebral vessel lysates from OVX rats 3 h after administration of IL-1. Animals were pretreated with either 20 mg/kg caffeic acid phenethyl ester (CAPE) or vehicle control [polyethylene glycol (PEG-400)] before the IL-1 injection. Protein bands migrating at 72 and 42 kDa were detected using antibodies directed against COX-2 and -actin, respectively. B: COX-2 densitometric data are illustrated as fold changes vs. vehicle-treated groups. Values are means ± SE; n = 3 rats; *P 0.05, significantly different from vehicle-treated group.

We therefore hypothesized that estrogen regulates cerebrovascular COX-2 induction by suppressing NF-B pathway activity. To test this hypothesis, NF-B p65 DNA-binding activity was measured in nuclear extracts of cerebral vessels isolated from OVX and OVX+E animals treated for 20 min with IL-1 or saline. As shown in Fig. 8, levels of NF-B p65 DNA binding were not different in cerebral vessels from OVX and OVX+E animals after saline treatment. Nuclear NF-B levels were also similar between saline-treated (OVX, 24 ± 6 vs. OVX+E, 16 ± 6%) and uninjected (OVX, 15 ± 4 vs. OVX+E, 12 ± 4%) animals. Interestingly, IL-1 injection significantly increased NF-B p65 DNA-binding activity in cerebrovascular tissue from both OVX and OVX+E animals compared with saline controls (Fig. 8). However, NF-B levels were significantly lower in cerebrovascular nuclear extracts from OVX+E vs. OVX animals treated with IL-1 (Fig. 8).

View larger version (36K): [in this window] [in a new window]   Fig. 8. Effects of estrogen treatment on NF-B p65 DNA-binding activity after IL-1 treatment. With the use of an immunoassay-based kit, NF-B p65 DNA-binding activity was measured in nuclear extracts from OVX and OVX+E cerebral vessels isolated 20 min after injection of saline or IL-1. Values are normalized to that of the positive control signal. Values are means ± SE; n = 3 or 4 rats; *P 0.05, significantly different.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inflammatory prostaglandins are important mediators of the vascular response to cerebral injury and are believed to contribute to the pathogenesis of ischemic stroke. In this study, we demonstrate that in vivo cytokine induction of the inflammatory COX-2 pathway in cerebral vessels is dramatically suppressed by estrogen treatment. In estrogen-deficient female rats, the endothelium of cerebral arteries responded to circulating IL-1 with expression of COX-2 protein that correlated with increased PGE₂ production and decreased vascular tone. This IL-1-mediated induction of COX-2 was dependent on NF-B activation. In contrast, after chronic estrogen replacement, cerebrovascular COX-2 expression, PGE₂ production, and cerebral vascular tone were all unaffected by IL-1 administration. Chronically circulating estrogen thus has potent anti-inflammatory effects with respect to the cerebral vascular response to IL-1 administration. Estrogen modulation of cerebrovascular inflammatory prostanoid pathways may contribute to the ability of female gonadal steroids to reduce brain damage after ischemia (24, 53).

IL-1 is one of the primary cytokines produced after ischemic brain insult (9). In our study, the cytokine was injected peripherally because this has been shown to induce COX-2 protein expression in cerebrovascular tissue (4). IL-1 caused an induction of COX-2 expression in cerebral vessels of the estrogen-deficient rat; a peak response occurred 3 h after treatment but persisted through 6 h. In arteries, COX-2 was localized in the endothelial cell layer. Using cultured cells, others have shown a similar time course of COX-2 expression after exposure to IL-1 (35, 51) as well as other cytokines (27). The present study focused on the effects of estrogen on the inflammatory response to IL-1; therefore, we compared estrogen-deficient with chronically estrogen-treated females. Additional studies are necessary to explore the effects of gender. However, preliminary studies suggest a similar correlation with estrogen levels in male and female rats as well as throughout the course of the estrus cycle (L. Sunday, S. P. Duckles, and D. N. Krause, unpublished observations). We demonstrated that induction of COX-2 by IL-1 was suppressed by prior treatment with estrogen. It is possible that PGE synthase is also induced by IL-1; however, additional studies are necessary to explore whether this enzyme is also suppressed by estrogen.

The ability of estrogen to suppress IL-1 induction of PGE₂ production persisted when MPA treatment was combined with estrogen treatment for 1 mo. MPA is a common ingredient of combination estrogen-progestin hormone-replacement therapy, and some have speculated that addition of MPA may negate the beneficial cardiovascular actions of estrogen. Our finding that MPA does not block the suppressive actions of estrogen on PGE₂ production is consistent with previous findings that MPA does not affect estrogen-induced increases in cerebrovascular eNOS (30) or suppression of ICAM-1 and E-selectin in cultured cells (32).

To better understand the physiological significance of inflammatory prostaglandin induction in the cerebral circulation, we measured contractile function of small-caliber branches of the middle cerebral artery. These were used because they are important regulators of cerebral vascular resistance and are situated downstream from the typical site of thromboembolic occlusion and thus contribute to postischemic hyperemia (51). Consistent with our previous studies (18, 37), we found that within the range of physiological pressures, saline-treated OVX rats displayed greater vascular tone than their estrogen-treated counterparts. Administration of IL-1 to OVX rats dramatically decreased isolated cerebral arterial tone, but there were no effects of IL-1 treatment on vascular tone in estrogen-treated animals. Neither estrogen nor IL-1 affected the pial artery autoregulatory response. These findings suggest that estrogen reduced IL-1-mediated endothelial-dependent cerebral vasodilation; this would perhaps be manifest as diminished hyperemia in the in vivo situation (23). Based on the results of others (3, 5, 31, 36), we suspected that the IL-1-mediated decrease in cerebrovascular tone was perhaps governed by COX-2-derived PGE₂. The endothelial localization of COX-2, the known vasodilatory effects of PGE₂ (14), and the decrease in PGI₂ after IL-1 treatment all support this hypothesis. As expected, the selective COX-2 inhibitor NS-398 had no effects on vascular tone of IL-1-exposed arteries from estrogen-treated rats. However, in arteries from OVX rats, NS-398 partially reversed the decrease in tone caused by IL-1. These results suggest the induction of additional vasodilators unrelated to COX-2. The most likely explanation is that IL-1 also induces inducible nitric oxide synthase expression, which would produce the potent vasodilator nitric oxide (9, 36, 40). In fact, preliminary studies from our laboratory suggest that this is the case (L. Sunday, S. P. Duckles, and D. N. Krause, unpublished observations).

An unanticipated effect was the slight decrease in tone observed when arteries from saline-treated OVX rats were exposed to NS-398. We found that COX-2 is present, albeit at low levels, in cerebral vessels of saline-treated OVX rats; this would result in the synthesis of prostaglandin endoperoxide (PGH₂) from arachidonic acid. Although PGH₂ is typically converted to any one of the prostanoids by prostaglandin-specific synthases, our work (37) as well as that of others (8) indicates that in the absence of estrogen, the activity of an endogenous thromboxane-PGH₂ receptor agonist speculated to be PGH₂ is upregulated. Blockade of such a constrictor substance by NS-398 could account for the decrease of tone in arteries from saline-treated OVX rats.

The potential interplay of multiple cell and tissue types in the intact organism complicates the mechanistic interpretation of this in vivo study. Differences in metabolism of circulating IL-1, however, do not account for the differential expression of COX-2 pathway activity because serum IL-1 levels were not affected by estrogen treatment. The expression of IL-1 receptors in cerebral vascular endothelium (15), the colocalization of COX-2 and IL-1 receptor mRNA in brain endothelial cells (4), and the rapid activation (20 min) of NF-B demonstrated here all suggest that IL-1 is directly responsible for the induction of inflammatory prostaglandins in cerebral vessels. Therefore, signaling mechanisms downstream to the activation of IL-1 receptors may be possible targets for estrogen regulation.

Activation of transcription by NF-B is critical in initiating the development of inflammatory processes. NF-B is a transcriptional activator that is normally maintained inactive in the cytosol while complexed with the inhibitory protein IB (28). Inflammatory stimuli such as IL-1 cause degradation of IB and translocation of NF-B dimers to the nucleus. There they bind B consensus sequences and activate target gene transcription. In cultured cells, IL-1 has been shown to induce the transcription of COX-2 via NF-B activation in some studies (5, 34) but not in others (50). Furthermore, the gene for COX-2 possesses two B sites in its promoter (34). In our study, we provide evidence that peripheral IL-1 treatment induces cerebrovascular COX-2 protein expression via the activation of NF-B.

In cell culture, estrogen inhibits the activation of NF-B by preventing IB degradation (44). Alternatively, estrogen might act through a mechanism unrelated to degradation of IB (17), for example, by inhibiting NF-B transport into the nucleus or reducing NF-B binding activity. In our study, estrogen only partially suppressed NF-B DNA-binding activity, whereas it completely inhibited IL-1 induction of COX-2 and PGE₂ and thus raised the possibility that estrogen also suppresses NF-B-dependent activation of COX-2 transcription. There is emerging evidence that the estrogen receptor complex, by utilizing transcriptional coactivators, can prevent NF-B from inducing gene expression even when the pathway has been activated (45). The fact that 17-estradiol, which is the inactive enantiomer of 17-estradiol, did not suppress the induction of PGE₂ by IL-1 supports the role of the estrogen receptor in mediating these effects.

The association of COX-2 products with the inflammatory sequelae of ischemic brain injury is well recognized. In fact, selective COX-2 inhibitors substantially reduce infarct volume (35) and neurological deficits (49) after middle cerebral artery occlusion in rodents. Reduction of PGE₂ accumulation in the infarcted brain of animals treated with COX-2 inhibitors has also been noted (35). Furthermore, COX-2-null mice display less damage than wild-type mice after experimental stroke (25). Together these observations suggest that inflammatory prostaglandins contribute to ischemic brain injury, and suppression of COX-2 function may confer protection against stroke. Vascular-derived prostanoids in particular may play an important part in cerebral inflammation after ischemic stroke as they may exert a driving role in the initiation of the inflammatory cascade. For example, COX-2 is induced in cerebral vascular tissue after injury (13). COX-2 is primarily associated with production of PGE₂ (22), although it can synthesize PGI₂ as well (1). The contribution of PGE₂ to the inflammatory cascade is underscored by its ability to upregulate cytokine expression (21) and induce ICAM-1 expression in cerebral endothelial cell cultures (46). Furthermore, the COX inhibitor indomethacin abrogates IL-1- and ischemia-mediated induction of VCAM-1, ICAM-1, and E-selectin in cultured cerebral endothelial cells (47). These findings suggest that PGE₂ may indirectly influence monocyte and polymorphonuclear leukocyte adhesion and transmigration out of the vasculature and into the central nervous system, which is a process believed to contribute to secondary injury after acute experimental stroke (16). Therefore, suppression of inflammatory prostaglandin production in the vasculature may protect the brain after injury.

We have demonstrated that chronic in vivo 17-estradiol treatment of ovariectomized rats almost completely inhibits the induction of cerebrovascular COX-2 and synthesis of PGE₂ when assessed 3 h after IL-1 injection. This observation has also been reported in microglia (52) and uterine fibroblasts (42). Whether COX-2 and PGE₂ are essential to the expression in the cerebral endothelium of cell adhesion molecules and whether this process is regulated by 17-estradiol remains to be elucidated. Interestingly, 17-estradiol suppresses cell adhesion molecule expression in cultured endothelial cells treated with cytokines (6, 17) and reduces leukocyte accumulation in cerebral venules in vivo (41).

In conclusion, systemic IL-1 induced NF-B-dependent expression of COX-2 in the ovariectomized rat cerebral circulation, and COX-2 appeared to localize to the endothelial cell layer. Chronic 17-estradiol treatment, however, inhibited IL-1 induction of COX-2 that was mediated in part by attenuation of NF-B activation. As a result, estrogen prevented the IL-1-induced upregulation of PGE₂ in an estrogen receptor-dependent manner regardless of whether MPA was present. In addition, 17-estradiol prevented the dramatic decrease in arterial tone that was observed after IL-1 exposure; however, suppression of COX-2 vasodilator pathways appeared to be only partially responsible for this effect. Although IL-1 and COX-2 pathways have been shown to contribute to ischemic brain injury, it remains to be determined whether suppression of cerebrovascular inflammatory prostanoid production can be added to the growing list of mechanisms by which estrogen acts as a neuroprotectant.

	ACKNOWLEDGMENTS

 
The authors thank Jonnie Stevens for performing surgeries and radioimmunoassays and Dr. Steven Hou (University of California Irvine Biological Sciences Imaging Facility) for assistance with confocal microscopy.

GRANTS

This project was supported by National Institutes of Health Grant R01 HL-50775 and by a Grant-in-Aid from the American Heart Association, National Center.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. P. Duckles, Dept. of Pharmacology, College of Medicine, Univ. of California, Irvine, CA 92697-4625 (E-mail: spduckle{at}uci.edu).

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Akarasereenont P, Techatrisak K, Chotewuttakorn S, and Thaworn A. The induction of cyclooxygenase-2 in IL-1-treated endothelial cells is inhibited by prostaglandin E₂ through cAMP. Mediators Inflamm 8: 287–294, 1999.[CrossRef][ISI][Medline]
2. Arribas SM, Vila E, and McGrath JC. Impairment of vasodilator function in basilar arteries from aged rats. Stroke 28: 1812–1820, 1997.[Abstract/Free Full Text]
3. Brian JE Jr, Moore SA, and Faraci FM. Expression and vascular effects of cyclooxygenase-2 in brain. Stroke 29: 2600–2606, 1998.[Abstract/Free Full Text]
4. Cao C, Matsumura K, Yamagata K, and Watanabe Y. Endothelial cells of the rat brain vasculature express cyclooxygenase-2 mRNA in response to systemic IL-1: a possible site of prostaglandin synthesis responsible for fever. Brain Res 733: 263–272, 1996.[CrossRef][ISI][Medline]
5. Catley MC, Chivers JE, Cambridge LM, Holden N, Slater DM, Staples KJ, Bergmann MW, Loser P, Barnes PJ, and Newton R. IL-1beta-dependent activation of NF-kappaB mediates PGE₂ release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett 547: 75–79, 2003.[CrossRef][ISI][Medline]
6. Caulin-Glaser T, Watson CA, Pardi R, and Bender JR. Effects of 17-estradiol on cytokine-induced endothelial cell adhesion molecule expression. J Clin Invest 98: 36–42, 1996.[ISI][Medline]
7. Davidge ST. Prostaglandin H synthase and vascular function. Circ Res 89: 650–660, 2001.[Abstract/Free Full Text]
8. Davidge ST and Zhang Y. Estrogen replacement suppresses a prostaglandin H synthase-dependent vasoconstrictor in rat mesenteric arteries. Circ Res 83: 388–395, 1998.[Abstract/Free Full Text]
9. Del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X, and Feuerstein GZ. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain responses to ischemia. Brain Pathol 10: 95–112, 2000.[ISI][Medline]
10. Del Zoppo GJ and Hallenbeck JM. Advances in the vascular pathophysiology of ischemic stroke. Thromb Res 98: V73–V81, 2000.
11. De Vries HE, Hoogendoorn KH, van Dijk J, Zijlstra FJ, van Dam AM, Breimer DD, van Berkel TJ, de Boer AG, and Kuiper J. Eicosanoid production by rat cerebral endothelial cells: stimulation by lipopolysaccharide, interleukin-1 and interleukin-6. J Neuroimmunol 59: 1–8, 1995.[CrossRef][ISI][Medline]
12. De Vries HE, Kuiper J, de Boer AG, van Berkel TJC, and Breimer DD. The blood brain barrier in neuroinflammatory diseases. Pharmacol Rev 49: 143–155, 1997.[Abstract/Free Full Text]
13. Domoki F, Veltkamp R, Thrikawala N, Robins G, Bari F, Louis TM, and Busija DW. Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries. Am J Physiol Heart Circ Physiol 277: H1207–H1214, 1999.[Abstract/Free Full Text]
14. Ellis EF, Wei EP, and Kontos HA. Vasodilation of cat cerebral arterioles by prostaglandins D₂, E₂, G₂, and I₂. Am J Physiol Heart Circ Physiol 237: H381–H385, 1979.[Abstract/Free Full Text]
15. Ericsson A, Liu C, Hart RP, and Sawchenko PE. Type 1 interleukin-1 receptor in the rat brain: distribution, regulation, and relationship to sites of IL-1-induced cellular activation. J Comp Neurol 361: 681–698, 1995.[CrossRef][ISI][Medline]
16. Frijns CJM and Kappelle LJ. Inflammatory cell adhesion molecules in ischemic cerebrovascular disease. Stroke 33: 2115–2122, 2002.[Abstract/Free Full Text]
17. Galea E, Santizo R, Feinstein DL, Adamsom P, Greenwood J, Koenig HM, and Pelligrino DA. Estrogen inhibits NFB-dependent inflammation in brain endothelium without interfering with IB degradation. Neuroreport 13: 1469–1472, 2002.[CrossRef][ISI][Medline]
18. Geary GG, Krause DN, and Duckles SP. Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries. Am J Physiol Heart Circ Physiol 275: H292–H300, 1998.[Abstract/Free Full Text]
19. Geary GG, Krause DN, and Duckles SP. Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms. Am J Physiol Heart Circ Physiol 279: H610–H618, 2000.[Abstract/Free Full Text]
20. Gerlag DM, Ransone L, Tak PP, Han Z, Palanki M, Barbosa MS, Boyle D, Manning AM, and Firestein GS. The effect of a T cell-specific NF-kappaB inhibitor on in vitro cytokine production and collagen-induced arthritis. J Immunol 165: 1652–1658, 2000.[Abstract/Free Full Text]
21. Hinson RM, Williams JA, and Shacter E. Elevated interleukin 6 is induced by prostaglandins E₂ in a murine model of inflammation: possible role of cyclooxygenase-2. Proc Natl Acad Sci USA 93: 4885–4890, 1996.[Abstract/Free Full Text]
22. Hurley SD, Olschowka JA, and O'Banion MK. Cyclooxygenase inhibition as a strategy to ameliorate brain injury. J Neurotrauma 19: 1–15, 2002.[CrossRef][ISI][Medline]
23. Hurn PD, Littleton-Kearney MT, Kirsch JR, Dharmarajan AM, and Traystman RJ. Postischemic cerebral blood flow recovery in the female: effect of 17-estradiol. J Cereb Blood Flow Metab 15: 666–672, 1995.[ISI][Medline]
24. Hurn PD and Macrae IM. Estrogen as a neuroprotectant in stroke. J Cereb Blood Flow Metab 20: 631–652, 2000.[CrossRef][ISI][Medline]
25. Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, and Ross ME. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc Natl Acad Sci USA 98: 1294–1299, 2001.[Abstract/Free Full Text]
26. Maffia P, Ianaro A, Pisano B, Borrelli F, Capasso F, Pinto A, and Ialenti A. Beneficial effects of caffeic acid phenethyl ester in a rat model of vascular injury. Br J Pharmacol 136: 353–360, 2002.[CrossRef][ISI][Medline]
27. Mark KS, Trickler WJ, and Miller DW. Tumor necrosis factor- induces cyclooxygenase-2 expression and prostaglandin release in brain microvessel endothelial cells. J Pharmacol Exp Ther 297: 1051–1058, 2001.[Abstract/Free Full Text]
28. McKay LI and Cidlowski JA. Molecular control of immune/inflammatory responses: interactions between nuclear factor-B and steroid receptor-signaling pathways. Endocr Rev 20: 435–459, 1999.[Abstract/Free Full Text]
29. McNeill AM, Kim N, Duckles SP, and Krause DN. Chronic estrogen treatment increases levels of endothelial nitric oxide synthase protein in rat cerebral microvessels. Stroke 30: 2186–2190, 1999.[Abstract/Free Full Text]
30. McNeill AM, Zhang C, Stanczyk FZ, Duckles SP, and Krause DN. Estrogen increases endothelial nitric oxide synthase via estrogen receptors in rat cerebral blood vessels: effect preserved after concurrent treatment with medroxyprogesterone acetate or progesterone. Stroke 33: 1685–1691, 2002.[Abstract/Free Full Text]
31. Miyamoto O, Tamae K, Kasai H, Hirakawa H, Hayashida Y, Konishi R, and Itano T. Suppression of hyperemia and DNA oxidation by indomethacin in cerebral ischemia. Eur J Pharmacol 459: 179–186, 2003.[CrossRef][ISI][Medline]
32. Mueck AO, Seeger H, and Wallwiener D. Medroxyprogesterone acetate vs. norethisterone: effect on estradiol-induced changes of markers for endothelial function and atherosclerotic plaque characteristics in human female coronary endothelial cell cultures. Menopause 9: 273–281, 2002.[CrossRef][ISI][Medline]
33. Natarajan K, Singh S, Burke TR Jr, Grunberger D, and Aggarwal BB. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-