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Am J Physiol Heart Circ Physiol 289: H1843-H1850, 2005. First published July 8, 2005; doi:10.1152/ajpheart.00465.2005
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Testosterone augments endotoxin-mediated cerebrovascular inflammation in male rats

Ali Razmara, Diana N. Krause, and Sue P. Duckles

Department of Pharmacology, School of Medicine, University of California, Irvine, California

Submitted 9 May 2005 ; accepted in final form 7 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of inflammatory mechanisms contributes to cerebrovascular pathophysiology. Male gender is associated with increased stroke risk, yet little is known about the effects of testosterone in the cerebral circulation. Therefore, we explored the impact of testosterone treatment on cerebrovascular inflammation with both in vivo and in vitro models of inflammation. We hypothesized that testosterone would augment the expression of two vascular markers of cellular inflammation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Using four groups of male rats [intact, orchiectomized (ORX), and ORX treated with either testosterone (ORXT) or the testosterone metabolite 17{beta}-estradiol (ORXE)], we determined effects of the sex hormones on cerebrovascular inflammation after intraperitoneal LPS injection. Western blot analysis showed that induction of inflammatory markers was increased in cerebral blood vessels from ORXT rats compared with intact or ORX rats. In contrast, in cerebral blood vessels from ORXE rats, there was a significant decrease in endotoxin-induced COX-2 and iNOS protein levels. Confocal microscopy of cerebral blood vessels from ORXT rats showed increased COX-2 and iNOS immunoreactivity in both endothelial and smooth muscle cells after LPS treatment. In vitro incubation with LPS also induced COX-2 in pial vessels isolated from the four animal treatment groups, with the greatest induction observed in ORXT vessels compared with the ORX and ORXE groups. Production of PGE2, a principal COX-2-derived prostaglandin end product, was also greatest in cerebral vessels isolated from ORXT rats. In conclusion, testosterone increases cerebrovascular inflammation; this effect may contribute to stroke differences between men and women.

estrogen; lipopolysaccharide; cyclooxygenase-2; inducible nitric oxide synthase

GIVEN THE RECENT CONTROVERSY surrounding use of hormone replacement therapy in women (38), testosterone therapy in elderly men also has come under scrutiny (35). Illicit use of androgens by younger men and athletes is a growing problem (36, 43). However, a lack of fundamental knowledge of the actions of sex hormones on nonreproductive organs hampers our ability to understand the short- and long-term consequences of androgen use. In addition, endogenous sex hormones are thought to contribute to sex differences in diseases such as stroke (1, 23, 42). Because of the critical role of the cerebral circulation in the pathogenesis of stroke, it is particularly important to determine whether gonadal hormones affect cerebrovascular function.

Relatively little is known regarding the effects of testosterone on vascular function in general (31), and there is even less knowledge concerning androgenic effects on the cerebral circulation. Because most studies focus on estrogen, the vascular effects of testosterone are comparatively unknown (39). We recently observed that chronic in vivo treatment with estrogen or testosterone affects cerebral artery reactivity in opposing ways: estrogen decreases vascular tone by enhancing production of endothelium-dependent vasodilators (33), whereas chronic testosterone treatment increases cerebrovascular tone (15). Physiological levels of testosterone increase vascular contractility mainly via actions on the endothelium (20, 23, 50), and this is also true in the cerebral circulation (15).

Inflammatory pathways play a central role in vascular dysfunction as well as in the pathophysiology of stroke-induced cerebral ischemia (9). After an ischemic stroke, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are expressed in response to inflammatory processes in many different areas of the brain and play key roles in ischemic damage (9). These inflammatory pathways may also be influenced by gonadal hormones (11, 47). We recently showed (32) that estrogen treatment of ovariectomized rats suppresses cerebrovascular increases in COX-2 protein levels after IL-1{beta} administration.

Testosterone and male gender are both associated with increased stroke risk (42), and testosterone treatment increases the size of ischemic lesions after middle cerebral artery occlusion in male rats (19). In contrast, estrogen treatment has been shown to decrease ischemic cerebral injury after experimental stroke in female animals (21, 48). Because the cerebral vasculature is an important site of cellular inflammation after ischemia, we examined the effects of testosterone treatment on the development of cerebrovascular inflammation in orchiectomized male rats. Because 17{beta}-estradiol is a downstream metabolite of testosterone with potentially opposite effects (23, 33), we also investigated whether 17{beta}-estradiol would affect cerebrovascular inflammation in males. As a model of cerebrovascular inflammation, we administered LPS, a bacterial endotoxin, and monitored the localization and production in cerebral blood vessels of two markers of vascular inflammation, COX-2 and iNOS. We hypothesized that testosterone would augment increases in the expression of COX-2 and iNOS in the cerebral vasculature of male rats after endotoxin-mediated inflammation. In contrast, consistent with our previous findings in female rats (32), we predicted that estrogen would suppress the induction of cerebrovascular COX-2 and iNOS in male rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal hormone treatments. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. We used four groups of male rats: intact, orchiectomized (ORX), ORX treated with testosterone (ORXT), and ORX treated with 17{beta}-estradiol (ORXE). Three-month-old male Fischer 344 rats (Charles River-SASCO Laboratories, Wilmington, MA) were orchiectomized while under anesthesia (46 mg/kg ketamine and 4.6 mg/kg xylazine ip). At the time of orchiectomy, two groups of ORX male rats were implanted subcutaneously at the base of the neck with Silastic capsules that contained either 17{beta}-estradiol (5 mm in length) or testosterone propionate (15 mm in length) (14, 15). After hormone treatment (4 wk), plasma testosterone levels were 3.5 ± 0.7 ng/ml in ORXT rats and plasma estrogen levels were 23 ± 8 pg/ml in ORXE rats. These plasma hormone levels were comparable to values measured in intact animals (14, 15). In ORX rats not receiving hormone implants, testosterone levels were below the level of detection (0.4 ng/ml) and estrogen levels were 9 ± 2 pg/ml.

At the time of surgery, all animals received an injection of penicillin (30,000 U, penicillin G benzathine-penicillin G procaine). After recovery from anesthesia, the animals were returned in individual cages to a vivarium (temperature-controlled room, 12:12-h light-dark cycle) with food and water ab libitum. The capsules were kept in place until the animals were euthanized.

Approximately 4 wk after surgery and hormone pellet implantation, animals were injected intraperitoneally with LPS from Salmonella typhosa (1 mg/kg; Sigma, St. Louis, MO) (18) or an equivalent volume of vehicle (0.9% saline ip). No mortality was observed after LPS administration. The four animal groups were euthanized at different time points up to 24 h after LPS or saline injections. The animals were anesthetized with CO2 and euthanized by decapitation, and whole brains were quickly removed, immediately placed on dry ice, and kept at –80°C until use. Blood samples were also collected by cardiac puncture for measurement of hormone levels by radioimmunoassay (Diagnostic Products, Los Angeles, CA).

Cerebral vessel isolation. Brains were thawed on ice, and cerebral vessels were isolated (26). Briefly, individual brains were gently Dounce homogenized in ice-cold 0.01 mol/l PBS (pH 7.4) and centrifuged at 3,000 g for 20 min at 4°C. The supernatant was discarded, and the pellet was washed several times by resuspension in PBS followed by centrifugation at 3,000 g. The pellet was resuspended in PBS, layered over a 15% dextran (mol mass 35–40 kDa; Sigma) density gradient solution, and finally centrifuged at 4,500 g for 45 min at 4°C. The aqueous supernatant was discarded, and the pellet (containing cerebral blood vessels) was collected over a 50-µm nylon mesh and washed with a strong stream of cold PBS. Vessels were collected from the nylon mesh with fine-tip forceps and stored at –80°C in PBS. Isolated cerebral vessels are a mixture of arteries, arterioles, veins, venules, capillaries, and their associated perivascular elements (26).

Tissue lysis and protein content determination. Blood vessel samples were lysed in a small glass homogenizer containing in (mmol/l) 50 {beta}-glycerophosphate, 2 MgCl2, 1 EGTA, 1 DL-dithiothreitol, and 1 phenylmethylsulfonyl fluoride, with 100 µmol/l NaVO3, 0.5% Triton X-100, 20 µmol/l pepstatin, 20 µmol/l leupeptin, and 0.1 U/ml aprotinin. Samples were then centrifuged at 4,500 g for 10 min at 4°C, and supernatants were collected. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Lysates were used immediately or stored at –80°C.

SDS-PAGE and Western blot analysis. Lysates were dissolved in Tris-glycine SDS sample buffer (containing {beta}-mercaptoethanol), and proteins were denatured by boiling for 5 min. For each Western blot experiment, vessel lysates from the four animal treatment groups were analyzed in a parallel manner. Equal amounts of sample protein (20 µg/lane) from each of the four animal groups were loaded together onto 8% Tris-glycine gels and separated by SDS-PAGE in a minigel apparatus. Broad-range biotinylated molecular mass markers (Bio-Rad) were loaded on each gel for protein band identification. After electrophoretic separation, protein was transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ). The membranes were blocked [6.5% nonfat dry milk in 0.01 mol/l PBS, 0.1% Tween 20 (T-PBS)] and incubated overnight at 4°C.

Membranes were then incubated with the primary antibody of interest: rabbit polyclonal anti-COX-2 (1:7,500 dilution; Cayman Chemical, Ann Arbor, MI), rabbit polyclonal anti-iNOS (1:1,000 dilution; Santa Cruz), or mouse monoclonal anti-{alpha}-actin (1:100,000 dilution; Sigma) in blocking buffer for 3 h at room temperature. Subsequently, the blots were rinsed five times for 5 min each in T-PBS. Membranes were then incubated with the appropriate secondary antibody: goat anti-rabbit IgG-horseradish peroxidase (1:5,000; Santa Cruz) or goat anti-mouse IgG-horseradish peroxidase (1:10,000; Santa Cruz) in blocking buffer for 1 h at room temperature. The membranes were then washed five times for 5 min each in T-PBS.

Protein bands were detected by electrochemiluminescence Western blotting detection reagents (Amersham) and then exposed to Hyperfilm (Amersham). Quantitation of band density was performed with the electrophoresis computer analysis program UN-SCAN-IT (Silk Scientific, Orem, UT). Levels of {alpha}-actin were used to confirm equal loading of the lanes.

Confocal microscopy. Cellular localization of COX-2 and iNOS was investigated with immunofluorescence labeling and scanning laser confocal microscopy as previously described (32, 41). Briefly, ORXT rats were injected with either vehicle or LPS, and 6 h later pial arteries were isolated under a microscope and cut into short segments. Isolated cerebral arteries were first dilated with papaverine (100 µmol/l) and then fixed with 3% formaldehyde for 30 min. The arteries were permeabilized with 0.1% Triton X-100 for 5 min and blocked in 1% bovine serum albumin-PBS for 1 h. After 4-h incubation at room temperature with anti-COX-2 (1:100 dilution; Cayman Chemical) or anti-iNOS (1:100 dilution, Santa Cruz) primary antibodies, the vessels were washed and then incubated with fluorescent secondary antibodies (10 µg/ml goat anti-rabbit Oregon green-488 and goat anti-rabbit Texas red; Molecular Probes) overnight at 4°C. After five 10-min washes in PBS, vessels were placed on microscope slides, covered with VectaShield mounting medium containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories) to counterstain nuclei, and coverslipped. Images were obtained with a Carl Zeiss Meta Laser Scanning Systems LSM 510 microscope with standard and UV lasers. Appropriate controls, such as secondary antibody alone, were used to assess nonspecific staining.

In vitro vessel incubation and PGE2 measurement. With a microscope, pial vessels were dissected from brains freshly isolated from each of the animal treatment groups. The collected vessels were then placed into mini-petri dishes containing 2.5 ml of DMEM (without L-glutamine or phenol red; Sigma) plus penicillin (10 IU/ml) and streptomycin (10 µg/ml). After 1-h equilibration at 37°C in 95% O2-5% CO2, vessels were transferred into mini-petri dishes containing 2.5 ml of either LPS-DMEM (100 µg/ml) (18) or DMEM vehicle controls. After 6-h incubation at 37°C in 95% O2-5% CO2, vessels were transferred into microfuge tubes containing 300 µl of fresh DMEM. After a further 30-min incubation at 37°C, the medium was collected and stored at –20°C. To measure PGE2 release, competitive enzyme immunoassay (Assay Designs) was carried out according to the manufacturer's protocol. After incubation, the vessels were lysed, and protein content was determined as described above to normalize PGE2 values to tissue protein concentrations. Western blots for COX-2 were then performed on the vessel lysates as described above.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with GraphPad Prism 4.0 software. Induction by LPS was determined in each experiment by subtracting the value obtained with the corresponding vehicle control from the value obtained with LPS treatment. Each Western blot included lysates from each of the four animal groups; therefore, differences in immunoblot optical density values were determined by ANOVA with repeated measures. One-way ANOVA or Student's t-test was also used where appropriate. Newman-Keuls post hoc analysis was used for pairwise comparisons. For all comparisons, statistical significance was set at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LPS induction of COX-2 and iNOS in cerebral vasculature. We initially investigated the time course of COX-2 and iNOS protein expression in cerebral blood vessels of intact, ORX, and ORXT male rats to determine peak LPS induction of these inflammatory markers. In Fig. 1, a representative Western blot of cerebral vessels from intact male rats treated with intraperitoneal LPS shows a characteristic double band for COX-2 protein migrating at 72 kDa, corresponding to the expected molecular mass of COX-2. The levels of COX-2 progressively increased, peaking at 6 h after LPS injection and decreasing afterwards. Figure 1 also shows a representative Western blot of cerebral vessels from intact male rats treated with LPS, demonstrating a band for iNOS protein migrating at 130 kDa. The band at 130 kDa correlates to the expected molecular mass of iNOS. Again, peak induction of iNOS was between 6 and 9 h after LPS injection. Control vessels from saline-treated rats exhibited undetectable or minimal amounts of COX-2 or iNOS proteins at 6 h after injection. These results indicate that peak induction of both COX-2 and iNOS in cerebral blood vessels occurred ~6 h after peripheral LPS injection. Therefore, to examine the hormonal effects on LPS induction of these inflammatory markers, the 6 h time point after LPS injection was used in subsequent experiments.



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  		Fig. 1. Time course of in vivo LPS induction of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins. Cerebral blood vessels were isolated from intact male rats euthanized at 1, 3, 6, 9, 12, or 24 h after intraperitoneal LPS administration. Representative Western blots of iNOS (130 kDa) and COX-2 (72 kDa) are shown. A similar time course was observed in orchiectomized (ORX) and testosterone-treated orchiectomized (ORXT) male rats.

 
COX-2 and iNOS localization in cerebral blood vessels after endotoxin induction. Laser scanning confocal microscopy was used to investigate the cellular localization of COX-2 and iNOS in cerebral blood vessels isolated from ORXT rats 6 h after peripheral LPS injection. Using the blue nuclear stain DAPI as a guide (2), we identified the endothelial cell layer by nuclei oriented in the direction of blood flow (Figs. 2A and 3A) and the smooth muscle layer by nuclei oriented perpendicular to the direction of blood flow (Figs. 2B and 3B). In vessels from saline-treated ORXT rats, no immunofluorescent staining for COX-2 (red) was detected in either endothelial (Fig. 2A) or smooth muscle (Fig. 2B) layers. However, intense COX-2 immunoreactivity (red) was observed in both endothelial (Fig. 2C) and smooth muscle (Fig. 2D) layers from cerebral vessels 6 h after LPS treatment. As shown in Fig. 3, A and B, there was no detectable staining for iNOS (green) in either endothelial (Fig. 3A) or smooth muscle (Fig. 3B) layers 6 h after saline treatment. Similar to findings for COX-2, significant immunofluorescence for iNOS was seen in both endothelial (Fig. 3C) and smooth muscle (Fig. 3D) layers 6 h after LPS treatment. We did not detect fluorescence in vessels from either saline- or LPS-treated animals when secondary antibodies were used in the absence of primary antibodies.



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  		Fig. 2. Laser scanning confocal microscopic localization of COX-2 in cerebral blood vessels isolated from ORXT rats injected with either saline or LPS 6 h before euthanasia: merged images of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei (blue) and immunofluorescence for COX-2 (red). A and B: endothelial (A) and smooth muscle (B) layers of a vessel after saline treatment. C and D: endothelial (C) and smooth muscle (D) layers of a blood vessel after LPS treatment.

 


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  		Fig. 3. Laser scanning confocal microscopic localization of iNOS in cerebral blood vessels isolated from ORXT rats injected with either saline or LPS 6 h before euthanasia: merged images of DAPI-stained nuclei (blue) and immunofluorescence for iNOS (green). A and B: endothelial (A) and smooth muscle (B) layers of a vessel after saline injection. C and D: endothelial (C) and smooth muscle (D) layers of a blood vessel after LPS treatment.

 
Hormonal modulation of in vivo endotoxin induction of COX-2 and iNOS. We then investigated hormonal effects on LPS induction of COX-2 and iNOS by comparing cerebral blood vessels from the four animal treatment groups. As mentioned above, the 6-h time point after LPS injection was chosen to examine enzyme induction. In all animal groups, the COX-2 and iNOS bands were weak or undetectable after injection of the saline vehicle (Figs. 4A and 5A). LPS treatment increased the expression of COX-2 (Fig. 4B) and iNOS (Fig. 5B) in cerebral blood vessels from intact, ORX, and ORXT rats; however, chronic testosterone treatment (ORXT) significantly augmented both COX-2 and iNOS protein levels compared with the other groups. In contrast, COX-2 and iNOS induction by LPS was markedly suppressed in ORXE rats (Figs. 4 and 5). The density of {alpha}-actin bands, run as a loading control, was constant in vessels from all treatment groups.



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  		Fig. 4. Effect of hormone treatments on LPS induction of COX-2 in vivo. A: representative Western blot shows COX-2 protein in cerebral blood vessels isolated from intact, ORX, ORXT, and 17{beta}-estradiol-treated ORX (ORXE) male rats. Animals were injected with saline vehicle (V) or LPS (L) 6 h before euthanasia. Bands migrating at 72 kDa were detected with an antibody directed against COX-2. Levels of {alpha}-actin migrating at 42 kDa are shown as protein loading controls. Levels of {alpha}-actin were not different between groups. B: densitometric analysis of the COX-2 bands is shown for the 4 animal groups treated with LPS. Values (expressed in optical density units) are means ± SE; n = 4 rats; ***P ≤ 0.05, significantly different relative to other 3 groups.

 


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  		Fig. 5. Effect of hormone treatments on LPS induction of iNOS in vivo. A: representative Western blot shows iNOS protein in cerebral blood vessels isolated from intact, ORX, ORXT, and ORXE male rats. Animals were injected with saline vehicle (V) or LPS (L) 6 h before euthanasia. Bands migrating at 130 kDa were detected with an antibody directed against iNOS. Levels of {alpha}-actin migrating at 42 kDa are shown as protein loading controls. Levels of {alpha}-actin were not different between groups. B: densitometric analysis of the iNOS bands is shown for the 4 animal groups treated with LPS. Values are means ± SE; n = 4 rats. ***P ≤ 0.05, significantly different relative to other 3 groups.

 
Hormonal modulation of in vitro endotoxin induction of COX-2. To test whether LPS acts directly on cerebral blood vessels, we examined the effects of prior in vivo hormone treatments on COX-2 induction after in vitro LPS exposure. As shown in Fig. 6A, levels of COX-2 protein in vessels from vehicle control incubations were quite low. These values were not different between the different animal groups: intact 26 ± 12, ORX 27 ± 12, ORXT 29 ± 12, and ORXE 28 ± 11 optical density units (P > 0.05). However, LPS incubation in vitro substantially induced COX-2 protein, and this induction differed significantly among the animal groups. Figure 6B shows optical density analysis of LPS-induced COX-2 protein levels after vehicle control levels were subtracted from each respective group. In vitro induction of COX-2 protein was significantly greater in cerebral vessels isolated from ORXT rats compared with vessels from intact, ORX, and ORXE rats. In contrast, in vitro LPS induction of COX-2 was significantly lower in vessels from ORXE rats compared with vessels from intact and ORX rats (Fig. 6B).



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  		Fig. 6. Effects of prior in vivo hormone treatment on in vitro induction of COX-2 by LPS. A: representative Western blot shows COX-2 protein levels in freshly dissected cerebral vessels incubated with either vehicle or LPS for 6 h at 37°C in vitro. Vessels were isolated from the pial surface of brains from intact, ORX, ORXT, and ORXE male rats. Bands migrating at 72 kDa were detected with an antibody directed against COX-2. Levels of {alpha}-actin migrating at 42 kDa are shown as protein loading controls. Levels of {alpha}-actin were not different between groups. B: densitometric analysis of COX-2 bands is shown for the 4 animal treatment groups. In all cases, the corresponding vehicle control value was subtracted from the LPS value and the difference, representing LPS induction, is shown. Values are means ± SE; n = 4 rats. ***P ≤ 0.05, significantly different relative to other 3 groups.

 
Hormonal modulation of in vitro endotoxin stimulation of PGE2 production. Because we observed that in vitro LPS treatment induced COX-2 protein and this was modulated by prior hormone treatment, we assessed whether in vitro LPS treatment would also stimulate the elaboration of a major COX-2-derived inflammatory prostaglandin, PGE2. Under control incubation conditions, we found no significant differences in PGE2 production among the vessels from the four animal treatment groups, consistent with our previous studies in females (32). However, in vitro incubation with LPS for 6 h increased PGE2 production by isolated vessels. For example, in ORX rats, LPS-stimulated PGE2 levels were 604 ± 165 pg/mg compared with 361 ± 103 pg/mg in vehicle controls (P ≤ 0.05). Similar differences were observed for the intact, ORXT, and ORXE groups. As shown in Fig. 7, after vehicle levels were subtracted from LPS-stimulated levels PGE2 release after in vitro LPS treatment was significantly greater in vessels from ORXT rats.



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  		Fig. 7. Hormonal effects on COX-2-derived PGE2 production after in vitro LPS treatment. Pial vessels isolated from intact, ORX, ORXT, and ORXE male rats were incubated with either DMEM as vehicle or LPS for 6 h at 37°C. Vessels were then placed in fresh medium for 30 min at 37°C. PGE2 released into the medium was measured by enzyme immunoassay and normalized to tissue protein. In all cases, the corresponding vehicle PGE2 value was subtracted from the LPS value and the difference, representing LPS stimulated PGE2 levels, is shown. Values are means ± SE; n = 4 rats. ***P ≤ 0.05, significantly different relative to other 3 groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the past decade, increasing evidence has pointed to the central role of inflammatory mechanisms in the development and progression of vascular disease. These include the pathogenesis of cerebral ischemia associated with subarachnoid hemorrhage, head injury, and stroke (4, 12, 28) as well as the pathophysiology of endothelial dysfunction and atherosclerosis (37). Interestingly, systemic infections, by triggering a systemic inflammatory response, have been linked to increased stroke risk (16, 44). Endothelial dysfunction has also been found in acute inflammation elicited by gram-negative bacterial LPS (34, 45, 46). Thus modulation of cerebrovascular inflammation by gonadal hormones has important implications for sex differences in disease as well as use of hormonal treatments.

During endotoxemia, underlying mechanisms that interfere with endothelial function include LPS-induced increases in proinflammatory cytokines (17). Similarly, among the earliest events after an ischemic insult are expression of proinflammatory genes, including upregulation of cytokines, chemokines, adhesion molecules, heat shock proteins, and other transcription factors (4). These cytokines increase expression of inducible proinflammatory enzymes, such as iNOS and COX-2, resulting in increased NO, inflammatory prostaglandins, and free radicals that contribute to the deleterious effects of inflammation (5, 27). The roles of COX-2 and iNOS in inflammation after experimental cerebral ischemia have been extensively reviewed (9). Interestingly, there is also evidence of interaction between iNOS and COX-2 in the postischemic brain, because iNOS-derived NO drives COX-2-derived production of toxic prostanoids and free radicals (30).

In the present study we used an endotoxin-induced inflammation model (10, 18) to investigate cerebrovascular inflammation. LPS is a proinflammatory component of the outer envelope of all gram-negative bacteria that targets the endothelium (3). Toll-like receptor (TLR)-4 is the well-known LPS receptor (6, 8), and endothelial cells have been shown to express this receptor (13). Moreover, TLRs seem to be central in the innate immune response of the central nervous system to a systemic bacterial infection (29), and TLR-4 is involved in this endotoxin-mediated cerebral inflammation response (22). We found that in vivo LPS administration markedly increased cerebrovascular inflammation, as indicated by increased levels of COX-2 and iNOS. Both endothelial and smooth muscle layers demonstrated significant COX-2 and iNOS immunoreactivity. We also used an in vitro organ culture of isolated cerebral vessels to investigate direct effects of LPS on the blood vessel. Interestingly, in vitro LPS treatment of isolated cerebral vessels increased levels of COX-2 protein as well as its enzymatic end product PGE2, supporting the notion that endotoxin induces a direct inflammatory response in cerebral blood vessels.

The major new finding of this study is that testosterone treatment exacerbates cerebrovascular inflammation. Chronic in vivo testosterone treatment significantly augmented the expression of COX-2 and iNOS in the cerebral vessels of male rats injected with endotoxin. Furthermore, prior testosterone treatment also resulted in substantially increased levels of COX-2 protein and PGE2 production in isolated pial vessels incubated directly with LPS. This finding demonstrates that cerebral vessels from testosterone-pretreated rats are qualitatively different and exhibit differential responses to endotoxin-mediated inflammation. These results support the notion that testosterone has a profound proinflammatory effect directly on the vascular bed of the brain.

Testosterone may exert its effects on the cerebral vasculature by means of its metabolites. Testosterone can be reduced to the more potent androgen 5{alpha}-dihydrotestosterone by 5{alpha}-reductase or aromatized to 17{beta}-estradiol by the action of aromatase (23). Thus we also examined the effects of estrogen in male rats. After estrogen treatment of male rats, induction of both COX-2 and iNOS after LPS injection was markedly attenuated in cerebral blood vessels. COX-2 induction by in vitro LPS incubation also was decreased in vessels isolated from ORXE animals. However, we did not detect a significant effect of prior estrogen treatment on PGE2 production in vitro. This may be a consequence of the relatively large variability in measurement of the small PGE2 production from ORXE vessels. We previously showed (32) in female rats that estrogen treatment suppresses induction of cerebrovascular COX-2 after injection of interleukin-1{beta}, a highly potent inflammatory cytokine. Preliminary studies in our laboratory also indicate that estrogen treatment of female rats significantly decreases LPS-induction of both COX-2 and iNOS (Sunday L, Krause DN, and Duckles SP, unpublished observations). These observed anti-inflammatory effects in the cerebral vasculature may contribute to the neuroprotective actions of estrogen (49) and may act to balance androgenic effects on inflammation in male rats.

It was shown previously that testosterone augments, whereas chronic estrogen attenuates, ischemic damage due to middle cerebral artery occlusion in male rats (19). Yet to our knowledge little is known about the impact of male sex steroids on cerebrovascular inflammation after stroke. Thus, in contrast to the actions of estrogen to suppress inflammation, testosterone appears to augment inflammatory responses. These differences may contribute to the well-known sex differences in stroke risk, morbidity, and mortality (1). Specifically, actions of gonadal hormones on cerebrovascular inflammation may play a role in the previously identified neuroprotective actions of estrogen (21) as well as in the ability of testosterone to exacerbate ischemic damage in animal models of stroke (19).

Our results indicate that the gonadal hormones estrogen and testosterone modulate inflammatory pathways in the cerebral vasculature of male rats. However, the molecular signaling mechanisms by which sex steroids exert these effects remains to be elucidated. NF-{kappa}B is a ubiquitously expressed family of transcription factors that are activated by nuclear translocation and regulate the transcription of proinflammatory genes in a wide variety of cell types (25). This transcription factor activates over 100 genes that are either directly or indirectly proinflammatory (24). Ospina et al. (32) showed that IL-1{beta} induction of COX-2 protein in the cerebral vasculature was dependent on NF-{kappa}B and in vivo estrogen treatment attenuated NF-{kappa}B activation. It has also been shown that estrogen inhibits NF-{kappa}B activation in cell culture (40). Furthermore, the potent androgen dihydrotestosterone increases vascular cell adhesion molecule-1 expression in male human endothelial cells via a NF-{kappa}B-dependent pathway (7). It will be essential to investigate the influence of testosterone on the TLR-4-mediated actions of LPS on translocation and activity of the critical proinflammatory NF-{kappa}B transcription factor, especially in light of possible interactions between the androgen receptor and NF-{kappa}B (25).

An important aspect of the biological actions of testosterone is its local tissue conversion to diverse bioactive metabolites, including dihydrotestosterone through 5{alpha}-reductase and estradiol through aromatase (23). It is likely that the balance of these two actions on the androgen and estrogen receptors may be important in the outcome of cerebral ischemia. However, the extent to which these two pathways of testosterone metabolism contribute to the role of endogenous hormones in males is still not known. Future studies of the actions of these bioactive metabolites using in vivo replacement models as well as enzyme and receptor inhibitors will be important to more fully understand the effects of testosterone on cerebrovascular function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Monetary support for this project was provided by National Heart, Lung, and Blood Institute Grant R01-HL-50775.


   ACKNOWLEDGMENTS
 
We thank Jonnie Stevens for performing the animal hormone replacement treatments and surgeries as well as Dr. Zifu Wang for assistance with confocal microscopy. Mariam Seddiqi also assisted with the Western blots.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. P. Duckles, Dept. of Pharmacology, School of Medicine, Univ. of California-Irvine, 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
 GRANTS
 REFERENCES
 

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