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a Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah 84322
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(6-keto-PGF1), the stable metabolite of prostacyclin, using an enzyme immunoassay. In addition, the location of PTGS-2 after LPS treatment was analyzed by immunohistochemistry in intact artery segments. Denuded arteries (endothelium absent) did not show increases in PTGS-2 protein in the homogenates or 6-keto-PGF1 in the culture medium after LPS exposure. In contrast, cut uterine arteries responded to LPS stimulation with a significant increase in PTGS-2 protein in homogenates and 6-keto-PGF1 in culture medium. Immunohistochemical staining for PTGS-2 was associated with both endothelial cells and vascular smooth muscle cells. These results suggest that while both endothelial cells and vascular smooth muscle cells are associated with PTGS-2, after LPS exposure it is the endothelial cells that are essential in uterine artery increases in PTGS-2 and prostacyclin in response to LPS stimulation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of mating often permits introduction of LPS-bearing bacteria into the uterus [11], and LPS has been shown to cause fetal death and abortion in animals [12–16]. One mode of action of LPS is the initiation of cellular events that activate phospholipase A2, which in turn releases arachidonate [17]. Released arachidonic acid is metabolized by prostaglandin-endoperoxide synthase (PTGS) (also known as cyclooxygenase) and yields prostacyclin and other prostaglandins [18]. Prostacyclin is released by endothelium and acts as a vasodilator of vascular smooth muscle [12, 19]. Inhibition of PTGS, a rate-limiting enzyme in prostaglandin production, reduces the effects of LPS, including pregnancy loss [1, 12]. There are two isoforms of PTGS, PTGS-1 and PTGS-2, which have similar activities but are encoded by different genes and have marked regulatory and functional differences [12]. For example, PTGS-1 is constitutively expressed in many tissues, including sheep uterine arteries [20], and is believed to be involved in the homeostatic maintenance of the body [12]. In contrast, PTGS-2 is believed to have a differentiative role in the cells and is highly inducible during inflammation and in response to LPS [12, 21]. PTGS-2 has been shown to be expressed in the vascular smooth muscle and myometrial epithelium of rats after stimulation with the immune response protein interleukin-1ß [22].
We have found that stimulation of uterine arteries with LPS results in a significant increase in the level of PTGS-2, but not PTGS-1, as determined by Western analysis [23]. The level of PTGS-2 protein within the uterine arteries after exposure to LPS was significantly correlated with the amount of 6-keto-prostaglandin F1 (6-keto-PGF1), the stable metabolite of prostacyclin, produced by these same uterine arteries. These results suggest that PTGS-2 is used by uterine arteries in response to pathological stimuli such as LPS to cause a release of prostacyclin, a potent vasodilator.
The objective of this study was to identify the cell population—endothelial cells or vascular smooth muscle cells—in uterine arteries that respond to LPS stimulation. Uterine arteries with or without endothelial cells were cultured in the presence of LPS and analyzed for the production of 6-keto-PGF1 in the medium and the protein level of PTGS-2 in artery homogenates. In addition, histological sections of intact uterine arteries collected after LPS exposure were analyzed by immunohistology in order to localize PTGS-2 to a particular cell population.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicular- and Luteal-Phase Ewes: Synchronization
Mixed white-faced Western range ewes (60–70 kg) were observed daily for signs of behavioral estrus using a vasectomized ram. Ewes (n = 8) exhibiting normal estrous cycles (16–18 days) were given 2 injections i.m., 5 mg each, of prostaglandin F2 (PGF2; Lutalyse [Pharmacia & Upjohn, Kalamazoo, MI]) 4 h apart. Ewes were monitored for behavioral estrus approximately 48 h after the first PGF2 injection. Ewes exhibiting estrus (Day 0) were randomly paired and assigned into two groups, follicular (Day 0, n = 4) or luteal (Day 10, n = 4). Eight days after the first sign of estrus for each pair (n = 4), the ewe assigned to the follicular group was given PGF2 as described above, and the ewe assigned to the luteal group was given saline (2 injections i.m., equivalent volume to that of PGF2, 4 h apart). It has been reported that this synchronization protocol [24] results in follicular-phase ewes showing estrus within 48 h after first injection, whereas luteal-phase ewes do not show estrus at this time. All follicular-phase ewes exhibited estrus on the morning they were killed, and all were killed within 2 h of estrus detection. Luteal-phase ewes were killed on the same day and did not exhibit estrus. Uterine arteries were collected as described below. This protocol results in larger large follicles (> 6 mm) in follicular animals and larger, more vascular corpora lutea in luteal animals [23].
Procurement of Arteries
Synchronized ewes were subjected to nonsurvival surgery using the following drugs purchased from MWI (Nampa, ID): preanesthesia—xylazine HCl (20 mg/ml) at 0.1–0.2 mg/kg BW along with acepromazine maleate (10 mg/ml) at 0.05–0.1 mg/kg, both given i.m.; anesthesia—ketamine HCl, 22–44 mg/kg BW, given i.v., repeated once if needed. At the end of the tissue harvesting, the animal was exsanguinated by severing the caudal aorta. The mesometrium of the uterus was rapidly excised and placed into sterile PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) as previously described [25]. Uterine arteries were dissected free of connective tissue, fat, and veins, and the arteries were rinsed free of blood. The exterior, but not the lumen, of the arteries was gently rinsed in PBS containing 1% penicillin-streptomycin to remove debris and blood. Uterine arteries were cut into 3 equal portions. One portion was cut into six 2-cm pieces, and each piece was placed into 2 ml sterile RPMI-1640 containing 1% penicillin-streptomycin (RPMI) with either 0, 1, or 10 µg/ml LPS (0111:B4; Sigma Chemical Co., St. Louis, MO) for 24 h at 37°C in a shaking water bath; treatments were run in duplicate. These samples were termed uterine artery intact (UA-I, n = 8). The remaining two pieces were opened longitudinally. In one of these pieces, the endothelium/tunica intima was gently scraped (3–4 times) using a curved-end spatula constructed specifically for this purpose. This piece was termed uterine artery denuded (UA-D, n = 7). This method results in a relatively pure preparation of vascular smooth muscle void of endothelial cells [25, 26]. The remaining cut-open piece was not treated any further and was termed uterine artery cut-open (UA-O, n = 7). Both UA-O and UA-D were cut into six 2-cm pieces, and each piece was placed into 2 ml sterile RPMI with either 0, 1, or 10 µg/ml LPS as described for UA-I. Media and supplements were purchased from Gibco (Gaithersburg, MD) as phenol red free and endotoxin free. Cultures were performed in serum-free conditions. After 24 h, supernatants were collected and stored at -20°C; tissue segments were weighed and then stored at -20°C in homogenizing buffer (PBS, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.1% ß-mercaptoethanol; Sigma). In addition, a small portion of each intact uterine artery (UA-I) was collected for immunohistology and fixed in 4% formaldehyde in sodium cacodylate buffer (0.1 M, pH 7.4).
Analysis of Supernatants for 6-Keto-PGF1
Culture medium from each artery preparation was diluted (1:10 000, UA-I; or 1:5000, UA-0, UA-D) and then analyzed for 6-keto-PGF1 by enzyme immunoassay (EIA) according to the manufacturer's protocol (Cayman, Ann Arbor, MI). Samples were analyzed in duplicate, and means were calculated for each treatment; these values were then divided by the weight of the tissue so that final numbers represented nanograms of 6-keto-PGF1/mg tissue wet weight produced during the 24-h culture period.
Preparation and Analysis of Tissue for Western Analysis
Uterine artery segments were solubilized by grinding in homogenization buffer (described above) and sonicated as previously described [20, 23, 26]. Connective tissue was removed from vessel preparations by centrifugation, and the protein concentration of samples was determined using a Lowry assay procedure (Bio-Rad, Hercules, CA). UA-O and UA-D from a single animal were represented on each blot. Equal concentrations of protein (50 µg/ml) from each sample were resolved on a 7.5% polyacrylamide gel with 0.1% SDS at 100 V for 2–3 h at room temperature before transfer onto Immobilon P (Millipore, Bedford, MA) membrane at 100 V for 2 h at 4°C using the Mini-Protean II system (Bio-Rad). The membrane was probed as described by Amersham (Arlington Heights, IL) using the enhanced chemiluminescence kit (ECL) and exposure to hyperfilm (15 min).
The primary antibody used to detect PTGS-2 was produced by Cayman and was used at a dilution of 1:4000; the secondary antiserum was a horseradish peroxidase-conjugated donkey anti-rabbit (Amersham) and was used at a 1:8000 dilution as previously described [23]. The PTGS-2 antibody was shown to be specific for PTGS-2 and, as previously reported, does not recognize PTGS-1 [23, 27].
Densitometric Analysis
The levels of PTGS-2 protein as detected by Western analysis were determined by scanning densitometer using an Alpha Imager (Alpha Inotech Corp., San Diego, CA). The validation of this protocol was determined by scanning blots of serial dilutions of the standard for PTGS-2, and has been previously reported [23]. The standard curve blot was linear within the range of protein levels used in this study (data not shown).
Immunohistochemical Analysis of Arteries
Cellular staining for PTGS-2 in arteries was determined using immunohistochemical analysis for PTGS-2. A Vectastain ABC Elite kit (Vector Labs., Burlingame, CA) and the rabbit polyclonal antibody against PTGS-2 described above were used. Briefly, fixed artery sections were embedded in paraffin and cut into 6-µm sections. After deparaffinization, the sections were incubated in 3% H2O2 in methanol for 20 min to quench endogenous peroxidase activity. Immunolocalization of PTGS-2 was accomplished by using the primary antibody at a 1:400 dilution in PBS containing 1% BSA for 1 h at room temperature. Indirect immunoperoxidase detection via avidin-biotin-peroxidase system (Vector-Elite) with the chromogen 3,3'-diaminobenzidine (Vector Labs.) was used to visualize the area of staining. Sections were counterstained lightly with Harris hematoxylin (Sigma). An isotype control primary antibody (rabbit polyclonal against fish connexon, generously supplied by Alice Dymerski, Antibody Laboratory, Utah State University Biotechnology Center) was included at the same protein concentration as the primary antibody on a serial section for each artery examined. The number of vascular smooth muscle cells staining for PTGS-2 in uterine arteries from 3 of the follicular and 3 of the luteal animals was determined by two separate individuals in a blind test, who counted three randomly selected fields (x40) of artery sections run in duplicates of each treatment (0, 1, and 10 µg/ml LPS). In addition, the number of negative-staining vascular smooth muscle cells was also counted in these same sections, and the percentage positive PTGS-2-staining cells was determined.
Statistical Analysis
All data collected from uterine arteries treated with LPS in vitro were examined by ANOVA. Analysis was performed initially to determine whether there was a phase (follicular vs. luteal) effect. Since there was no phase effect, data were pooled across phase and examined by ANOVA using a completely randomized design with split-plot arrangements of in vitro treatments. Treatment (UA-I, UA-O, UA-D) was the whole-plot effect, and the dose of LPS (0, 1, 10 µg/ml) the subplot effect. Means were separated using single degree of freedom orthogonal contrasts to test for the effects of LPS addition (0 µg/ml LPS vs. 1 and 10 µg/ml LPS), LPS level (1 µg/ml LPS vs. 10 µg/ml LPS), and the interactions of LPS addition and LPS level with treatment (UA-I, UA-O, UA-D). Data for PTGS-2 protein levels as determined by Western analysis were log10 transformed in order to equalize variances prior to ANOVA. Differences in the number of vascular smooth muscle cells staining for PTGS-2 after LPS treatment, as determined by immunohistochemistry, were examined by one-way ANOVA. Means were separated by least-significant differences.
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RESULTS |
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There was no phase (follicular vs. luteal) effect (p > 0.32) on 6-keto-PGF1 production by uterine arteries. LPS addition had a significant (p < 0.001) effect on 6-keto-PGF1 production. This response appeared to be maximal at 1 µg/ml LPS, as the 10 µg/ml LPS level did not alter (p = 0.17) 6-keto-PGF1 production by arteries from the 1 µg/ml LPS level (Fig. 1). There was a significant LPS x tissue interaction (p = 0.006). The LPS addition x tissue interaction was explored by single degrees of freedom orthogonal contrasts because there was no effect of the LPS level (1 vs. 10 µg/ml LPS). Addition of LPS had a similar (p = 0.12) effect for UA-I and UA-O on production of 6-keto-PGF1 in response to LPS (Fig. 1); LPS enhanced production in these arteries. However, the effect of LPS addition was different for UA-I and UA-D (p = 0.003) and UA-O and UA-D (p = 0.022); UA-D failed to respond to LPS with an increased production of 6-keto-PGF1.
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PTGS-2 Protein Levels in Uterine Artery Homogenates
PTGS-2 was identified in uterine artery homogenates at an approximate molecular mass of 72 kDa and was in doublet form all of the time. There was no phase (follicular vs. luteal) effect (p = 0.56) on PTGS-2 production for the various artery preparations (UA-O and UA-D). A significant difference (p = 0.016) in the level of PTGS-2 protein was found in UA-O compared to UA-D. On average, LPS addition (0 µg/ml LPS vs. 1 and 10 µg/ml LPS) resulted in a significant increase (p = 0.002) in PTGS-2 protein, while LPS level (1 µg/ml vs. 10 µg/ml LPS) did not result in an additional increase in PTGS-2 protein found in uterine artery homogenates (p = 0.29). There was a significant (p = 0.001) LPS addition x tissue interaction; addition of LPS resulted in increases of PTGS-2 protein levels in UA-O but not UA-D homogenates (Fig. 2).
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Immunohistochemical Analysis of Arteries
Positive staining for PTGS-2 was associated with both endothelial cells and vascular smooth muscle cells in all uterine arteries treated with LPS. Neither arteries treated with 0 µg/ml LPS nor those stained with IgG controls exhibited positive staining of either endothelial cells or vascular smooth muscle cells (Fig. 3). After LPS exposure, all endothelial cells appeared to stain for PTGS-2. In contrast, a heterogeneous population of vascular smooth muscle cells staining for PTGS-2 existed after LPS exposure. There was an increase (p < 0.05) in the number of cells staining for PTGS-2 after LPS addition, and this increase continued with changes in LPS level (p < 0.05) (Fig. 4). The percentage (± SEM %) of vascular smooth muscle cells staining for PTGS-2 in uterine arteries treated with 0, 1, and 10 µg/ml LPS was 0.78% (± 0.52%), 35% (± 5.87%), and 48% (± 6.20%), respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in response to LPS. In contrast, uterine arteries that were denuded of endothelial cells did not show an enhanced production of 6-keto-PGF1 following LPS exposure. These results suggest that endothelial cells are essential in uterine artery LPS response; the findings are also consistent with endothelial cells as a primary target of LPS during endotoxemia [5–7]. The integrity of the artery (compare intact vs. cut-open) did not significantly alter the ability of these arteries to respond to LPS.
As seen with 6-keto-PGF1, PTGS-2 increased in uterine arteries with intact endothelium after LPS exposure, an observation consistent with the known inducible character of PTGS-2 [12, 21] and with our previously reported data [23]. In contrast, PTGS-2 did not increase in uterine arteries denuded of endothelial cells, suggesting that endothelial cells are important in the LPS-inducible PTGS-2 response by uterine arteries.
Immunohistology showed that the cell populations in the uterine artery that responded to LPS with an increase in PTGS-2 were both the endothelial cells and the vascular smooth muscle cells. Immunohistologically, PTGS-2 protein has been identified in the caruncular epithelium from Day 12 sheep placenta and in the intercaruncular glands of the Day 15 sheep placenta [28]. At term, PTGS-2 immunostaining occurred in the glandular epithelial cells of the sheep uterus and in a heterogeneous population of endometrial glands [28, 29]. No staining for PTGS-2 was detected in the stromal endometrium or myometrium of the pregnant sheep uterus [28]. Immunohistochemical staining of mouse uteri during the periimplantation period revealed PTGS-2 to be located primarily in the luminal epithelial and subepithelial stromal cells surrounding the blastocyst, as well as the decidual cells of the mesometrial pole [30, 31]. In the rat from Day 18 to parturition, PTGS-2 was localized primarily to the epithelial cells of the endometrium and smooth muscle cells in the circular layers of the myometrium, as well as epithelial and smooth muscle cells of the cervix [22]. In the present study, all endothelial cells in uterine arteries stained for PTGS-2 after exposure to 1 µg/ml LPS. However, the number of vascular smooth muscle cells staining for PTGS-2 increased with increasing doses of LPS. Some cells remained unstained for PTGS-2 at the maximal (10 µg/ml) dose of LPS used in this study. These data suggest that vascular smooth muscle cells are more heterogeneous in their response to LPS than the endothelial cells. The heterogeneous nature of vascular smooth muscle cells has been reported [32] and is especially prevalent following injury [33]. It appeared that vascular smooth muscle staining for PTGS-2 was more intense in cells in close proximity to the endothelial cells and on the outer edges of the artery segments, suggesting that there is an increased signal, possibly from endothelial cells, for increases in PTGS-2 after LPS exposure. Data from the EIA and Westerns showed that without the presence of endothelial cells, vascular smooth muscle cells were incapable of responding to LPS with increases in 6-keto-PGF1 or PTGS-2. It is unclear whether this dependence on endothelial cells by vascular smooth muscle cells is contact dependent or contact independent. Experiments are under way to examine more closely the nature of the dependency.
Immunohistochemistry showed that both endothelial cells and vascular smooth muscle cells stained for PTGS-2 after LPS exposure. However, the uterine artery culture data in the present experiment suggest that it is the endothelial cells, not the vascular smooth muscle cells, that are essential in uterine artery increases in prostacyclin and PTGS-2 in response to LPS exposure. These data support the hypothesis that the most effective treatments for endotoxemia would be those that target endothelial cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 435 797 2118; kvagnoni{at}cc.usu.edu
Accepted: April 6, 1999.
Received: December 3, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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concentrations. Biol Reprod 1994; 50:1297–1302.[Abstract]This article has been cited by other articles:
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 D. A. Habermehl, M. A. Janowiak, K. E. Vagnoni, I. M. Bird, and R. R. Magness Endothelial Vasodilator Production by Uterine and Systemic Arteries. IV. Cyclooxygenase Isoform Expression During the Ovarian Cycle and Pregnancy in Sheep Biol Reprod, March 1, 2000; 62(3): 781 - 788. [Abstract] [Full Text]
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