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Department of Obstetrics and Gynecology, Division of Gynecological Endocrinology and Reproductive Medicine (W.T., F.S., W.D., C.S., J.C.H.); Institute of Vascular Biology and Thrombosis Research (Z.Z.); Institute of Forensic Medicine (T.S., W.V.); and Institute for Tumor Biology and Cancer Research (T.W.), University of Vienna Medical School, General Hospital, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Walter Tschugguel, M.D., Department of Obstetrics and Gynecology, Division of Gynecological Endocrinology and Reproductive Medicine, University of Vienna Medical School, General Hospital, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail walter.tschugguel{at}akh-wien.ac.at
Abstract
Carbon monoxide, a gaseous activator of soluble guanylyl cyclase formed by a subtype of the enzyme heme oxygenase designated heme oxygenase-2 in vascular endothelium, has been found to dilate blood vessels independently from nitric oxide. Because of the parallels between nitric oxide and carbon monoxide, we speculated that estrogen might affect carbon monoxide production in vascular endothelium. Endothelial cells of human origin (umbilical vein and uterine artery) were incubated for 4 or 24 h with 10-12–10-6 M 17ß-estradiol. 17ß-Estradiol, at a concentration such as that attained during the ovulatory phase of the menstrual cycle (10-10 M), administrated for 4 h led to a 2-fold increase in intracellular carbon monoxide production and heme oxygenase-2 protein levels (P < 0.05). A reporter assay, measuring the formation of cGMP as the direct product of carbon monoxide-induced activation of soluble guanylyl cyclase in endothelial cells, also revealed a 56% increase in cellular cGMP after treatment with 10-10 M E2 17ß-estradiol (P < 0.05). By contrast, higher 17ß-estradiol concentrations had no significant respective effects due to nitric oxide synthase inhibition of carbon monoxide release. This 17ß-estradiol effect appeared to be ER dependent, as preincubation with tamoxifen (10-6 M) blocked the stimulatory effect of 17ß-estradiol in each instance. Our preliminary data indicate a potential role for carbon monoxide as a biological messenger molecule in estrogen-mediated regulation of vascular tone.
THE INCIDENCE OF cardiovascular disease, the leading cause of mortality in western societies, is higher in men than in premenopausal women, but increases in postmenopausal women (1). An abundance of epidemiological data supports a role for estrogens in this atheroprotective effect, prompting recommendations for their widespread use in postmenopausal replacement therapy (2).
However, the mechanism by which estrogen mediates vasoprotection remains to be fully clarified. It is traditionally thought to be due to potentially favorable changes in blood lipids and lipoproteins (3), but a number of human (4, 5) as well as animal studies strongly suggest a direct effect on the vascular system (6, 7) acting via vascular ER (4, 5).
17ß-Estradiol (E2) accounts for the endothelial expression of rate-limiting enzymes in the biosynthesis of the two important vasodilators, prostacyclin and nitric oxide (NO). E2 potentiates the effect of endothelin-1 on prostacyclin production in human umbilical vein endothelial cells (HUVECs) (8) and causes increased transcription of the endothelial nitric oxide synthase (eNOS) gene (9) as well as activation of eNOS (10) and NO release (11) in endothelial cells via nongenomic mechanisms.
Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation in mammals (12). It decomposes heme into biliverdin and releases free iron and carbon monoxide (CO). To date, three isoforms of HO have been characterized: HO1, HO2, and HO3 (13, 14). HO1 is widely expressed and is inducible by a host of stimuli that produce oxidative stress (15). On the other hand, HO2 occurs in neuronal populations and vascular endothelial cells (16), and the only known inducer of HO2 is adrenal glucocorticoid (17). HO3 has recently been identified in the rat heart, kidney, brain, and liver (14). Both vascular endothelium and smooth muscle express heme oxygenases (16, 18), and HO-catalyzed formation of CO has been documented in blood vessels (19). Upon endothelial release, CO diffuses to the underlying vascular smooth muscle cells to elicit relaxation by increasing cGMP production independently from NO (18, 20, 21), to cause hyperpolarization by opening potassium channels (22), and to decrease the production of vasoconstrictors such as endothelin (23).
NO is a well established effector molecule of E2-mediated vasoprotection. Because of the apparent parallels between NO and CO, we sought to clarify preliminarily whether at physiological levels E2 modulates CO production, HO2 protein and mRNA levels, and levels of cGMP, a direct product of NO- and CO-induced activation of soluble guanylate cyclase in HUVEC and human uterine artery endothelial cells (HAUEC) in both the presence and absence of estrogen and antiestrogen.
Materials and Methods
Endothelial cell culture
HUVEC and HAUEC were isolated from female umbilical veins and hysterectomized uteri, respectively as previously described (24). Cells were passaged on gelatin-coated 25-cm2 flasks (Costar, Cambridge, MA) in phenol red-free medium 199 (Sigma, St. Louis, MO) containing 20% heat-inactivated FCS (HyClone Laboratories, Inc., Logan, UT) and supplemented with penicillin (1000 IU/ml), streptomycin (1 mg/ml), fungizone (25 µg/ml; penicillin-streptomycin-fungizone solution, JRH Biosciences, Lenexa, KS), endothelial cell (EC) growth supplement (50 µg/ml; Technoclone, Vienna, Austria), and heparin (5 IU/ml; Hoffman-LaRoche Inc., Basel, Switzerland). Cells were used within four passages and were identified as endothelial by their characteristic cobblestone morphology, the presence of factor VIII antigen, and uptake of acetylated low density lipoprotein. Forty-eight hours before the experiments, heparin and endothelial cell growth supplement were removed from the medium. ECs were treated with E2 (Sigma) over a range of concentrations and time points, as indicated in Results and the figure legends. The role of ER in the response to E2 was determined during incubations performed in the simultaneous presence of 10-6 M tamoxifen (TAM; Sigma) added 1 h before E2. To examine the role of the NOS system on endothelial CO release, different dilutions of the nonspecific NOS inhibitor NG-nitro-L-arginine-methyl-ester (L-NAME; Sigma) were added to cells 30 min before E2 treatment.
CO detection system
CO release by vascular ECs was determined by spectrophotometric detection of CO-hemoglobin (25) in cell culture supernatants. In brief, supernatant was collected from confluent cells at the end of the incubation period. The percentage of COHb was determined spectrophotometrically using a U-3000 spectrophotometer (Hitachi, Tokyo, Japan), and the amount of pure CO was calculated and expressed as micrograms per liter total supernatant.
Western blotting
For cytosolic fractions, cells were lysed in Nonidet P-40 lysis buffer and further processed as previously described (26). In brief, the concentration of protein in aliquots of the lysates was measured with a bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL). Twenty micrograms of cellular extracts were used for experiments, and 5 µg of a rat spleen or a rat brain microsomal preparation (Chemicon, Temecula, CA) were used as positive controls for the detection of HO1 or HO2 protein, respectively. After quantification, proteins were electrophoresed through the use of standard SDS-PAGE on 8–18% gradient gels and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were blocked in a solution consisting of 5% nonfat dry milk and 5% BSA (Sigma) in 0.1 mol/liter Tris-HCl, 0.15 mmol/liter NaCl, and 0.01% Nonidet P-40, pH 7.5. Immunoreactions were performed with an anti-HO1 or anti-HO2 polyclonal antibody (StressGen, Victoria, Canada; dilution of both antibodies, 1:2,000). An appropriately diluted polyclonal rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) instead of the primary AB was used as a negative control for each AB. This was followed by a horseradish peroxidase-conjugated goat antirabbit IgG (Pierce Chemical Co.; dilution, 1:20,000). Specific reaction products were detected by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Quantitative real-time RT-PCR
Total RNA was extracted from cultured cells as previously described (27). Briefly, total RNA was prepared by ultracentrifugation of a guanidine-isothiocyanate lysate through a cesium trifluoroacetate cushion. Precipitated RNA was removed, diluted with absolute ethanol, pelleted by centrifugation, washed with 70% ethanol, dissolved in ribonuclease-free water, and stored at -80 C. The integrity of RNA was assessed by agarose gel electrophoresis. RT was performed on 500 ng total RNA using 200 U Moloney murine leukemia virus reverse transcriptase (ViennaLab Labordiagnostika GmbH, Vienna, Austria) and a commercially available reagent kit (Random Primed RT-Mix, ViennaLab Labordiagnostika GmbH). Synthesized cDNA was stored in aliquots at -80 C.
The amplification primers and the TaqMan probe for the HO2 real-time
PCR (Table 1
) resulting in a 71-bp
amplicon (position 841–911) (28) were designed with
Primer-Express software (PE Biosystems, Foster City, CA).
6-Carboxyfluorescein was used as the reporter dye, and
6-carboxytetramethylrhodamine was used as the quencher dye.
Oligonucleotide synthesis and purification were performed by
VBC-Genomics Bioscience Research GmbH (Vienna, Austria). The reaction
was carried out in a 25-µl total volume containing 2 µl cDNA, 25
pmol of each amplification primer, 5 pmol probe, and 12.5 µl 2
x TaqMan Universal Mix (PE Applied Biosystems). The
reaction conditions were 50 C for 2 min, 95 C for 10 min (activation of
the AmpliTaq-Gold polymerase), and then 40 cycles of 15 sec at 95 C
(denaturation), followed by 60 sec at 60 C (annealing and extension).
To correct variations linked to differences in the amount of RNA taken
for the reaction or to different levels of inhibition during RT or PCR,
we normalized HO2 expression using the glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene, a ubiquitously expressed housekeeping gene,
as a reference gene. The expression of this housekeeping gene was
quantified with the GAPDH Control Reagents Kit from PE Applied Biosystems according to the manufacturer’s guidelines. All HO2
and GAPDH experiments were carried out in triplicate, and several
negative controls were included. Fluorescence emission was continuously
monitored and analyzed by a GeneAmp 5700 Sequence Detection System
(PE Applied Biosystems) with GeneAmp 5700 SDS software
(version 1.1).
|
For the generation of HO2 and GAPDH standard curves, we used 2-fold serial dilutions of a HUVEC cDNA sample showing the highest HO2 mRNA expression levels in HO2 real-time RT-PCR as templates. Standard curves were constructed and calculated using GeneAmp 5700 SDS software by plotting the threshold cycle (PCR cycle at which a specific fluorescence becomes detectable) vs. the log of the cDNA starting quantity of each dilution step. These standard curves allowed us to interpolate the unknown HO2 and GAPDH mRNA expression levels in each analyzed sample.
cGMP measurement
HAUEC were cultured under standard culture conditions as described above, incubated with 3-isobutyl-1-methylxanthine (Sigma; final concentration, 0.5 mM) to inhibit phosphodiesterase activity and with the nonselective NOS inhibitor L-NAME (final concentration, 0.1 mM) to avoid demonstrating a potential effect of NO on cGMP levels. Both inhibitors were administrated 30 min before incubation with E2. Cells were solubilized in lysis buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.4), and 1% Triton X-100 (Sigma)], and 10 mM EDTA. Cell fragments were collected by centrifugation at 10,000 x g. Quantification of intracellular cGMP levels in cell lysates was assessed using cGMP RIA (Amersham Pharmacia Biotech) according to the supplier’s instructions, including an acetylation step. The protein content of the lysates was assessed using a Lowry-based protein assay (DC protein assay, Bio-Rad Laboratories, Inc., Richmond, CA). Duplicate measurements were performed on all samples.
Statistical analysis
The values are expressed as the mean ± SD. A preliminary analysis revealed that the datasets conformed to a normal distribution. Comparisons between groups were made using the exact version of the Wilcoxon test in SAS (SAS/STAT, 1989) (29). Adjustment for multiple testing was performed using the Bonferroni-Holm procedure (30). Statistical significance was defined as P < 0.05.
Results
Effect of E2 on CO levels
Four-hour incubation of HUVEC and HAUEC with
E2 increased CO levels in the cell culture
supernatant in a biphasic pattern, from 15 and 24 µg/liter at
baseline to 32 and 39 µg/liter at
10-10 M
E2, respectively (P < 0.05),
whereas TAM pretreatment fully blocked this effect in both instances
(Fig. 1, A and C). Neither 1 h (not
shown) nor 24 h (Fig. 1B) of E2 treatment
affected the levels of CO released.
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, preincubation of
HAUEC with a high L-NAME concentration (10 mM)
elicited an E2-dependent increase in CO release
at 10-10 M
E2, which remained elevated at
10-6 M
E2 vs. baseline (P <
0.05), whereas lower concentrations of L-NAME
(0.1 mM) had no significant effect on CO
levels.
|
Using an antibody specific for detection of HO1 protein with an
approximate molecular mass of 32 kDa, HUVEC were shown to be negative
for HO1 protein (Fig. 3). By contrast,
using a specific HO2 antibody that detects a 36-kDa HO2 protein, HUVEC
were positive for HO2 protein, even under untreated conditions (Fig. 3). Furthermore, similar to CO levels, 4-h incubation of HUVEC and
HAUEC with E2 induced HO2 protein levels in a
biphasic pattern (Fig. 3), reaching significance at
10-10 M
E2 (P < 0.05), showing 96% and
120% increases compared with incubation of cells with vehicle,
respectively (Fig. 4). TAM pretreatment
fully prevented the E2-mediated increase in HO2
protein levels (Figs. 3 and 4), demonstrating a receptor-dependent
mechanism of E2-mediated increase in HO2 protein
in those cells. However, at 1 h (not shown) or 24 h (Figs. 3 and 4B) of treatment, E2 did not change the
levels of HO2 protein.
|
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After 1 h of treatment, E2 induced HO2
mRNA expression in HUVECs in a biphasic pattern, reaching significance
only at a concentration of
10-10 M
E2 (P < 0.05), showing a 17%
increase compared with the control value (ethanol, <0.1%, used as a
vehicle for E2; Fig. 5). To ascertain ER involvement in the
noted E2 response, HUVECs were incubated with
10-6
M of the partial ER antagonist TAM 1 h
before E2 treatment. TAM pretreatment fully
prevented E2-stimulated HO2 gene expression (Fig. 5), whereas TAM alone, used to evaluate a potential agonistic effect of
this compound, showed no effect on HUVEC HO2 mRNA expression (not
shown). These data clearly demonstrate that E2 at
a concentration such as that attained during the ovulatory phase of the
menstrual cycle up-regulates HO2 mRNA levels in HUVEC by a
receptor-dependent mechanism. However, at 4 h (Fig. 5) or 24
h (not shown) of treatment, E2 did not change the
levels of HO2 mRNA.
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In subsequent experiments the functional significance of increased
cellular CO production was assessed by measuring the intracellular
formation of cGMP, a direct product of NO- and CO-induced activation of
guanylate cyclase. Experiments were performed after NOS inhibition to
avoid potential interactions between the NO and CO systems (12, 31). Four-hour incubation of HAUEC with
10-10 M
E2 increased cGMP levels from 1.8 to 2.8
fmol/1.5 x 103 cells (56% increase from
baseline; P < 0.05). This effect of
E2 was fully abolished by pretreatment of cells
with TAM (Fig. 6). However, after
stimulation of cells with higher E2
concentrations, cGMP levels returned to baseline (Fig. 6).
|
The mechanisms by which E2 exerts direct protective effects on the vasculature are incompletely understood. In the present preliminary study we focus on a novel pathway introducing CO as a potential new candidate mediating atheroprotective effects of E2. From the amount of CO detected in the medium, we first found that E2-treated endothelial cells of different human origins (HUVEC and HAUEC) released more CO in their culture medium than control cells. The required duration of E2 exposure was 4 h at a concentration such as that attained during the ovulatory phase of the menstrual cycle, and the effect could be inhibited by the E2 antagonists TAM (shown here) and ICI 182,780 (preliminary observations). These data suggest a traditional mechanism of estrogen action involving the functional ERs that have been characterized in this population of cells (32, 33). However, to our knowledge no estrogen-responsive elements or at least half-palindromic sites thereof have been reported in the promoter region of the HO2 gene, suggesting a regulation of CO release by E2 at the level of protein synthesis rather than at the gene level. We therefore explored the endothelial HO2 protein and mRNA.
HO2 protein levels in cells followed a similar course by estrogen treatment. These results support an enhancement by E2 on the protein level of HO2. This would probably occur during protein synthesis or even gene transfer. Quantitative real-time RT-PCR confirmed results at the gene level, except the effect was less pronounced and occurred at 1 h, but not 4 h, of stimulation. From the fact that increased HO2 transcription precedes increased protein levels, it is tempting to speculate that mRNA has been lost in the presence of the more stable protein. The less pronounced effect on the gene (17% increase in HUVEC) compared with the protein level (96% and 120% increase for HUVEC and HAUEC, respectively) further suggests that parts of the HO2 regulation may occur at the protein level independent of gene regulation.
We further observed that E2 elicits CO release with a biphasic concentration dependence, where CO release peaked (32 and 39 µg/L CO, corresponding to 1.1 and 1.3 µmol/L CO) at ovulatory E2 levels, but decreased at higher E2 levels. These effects of E2 are confirmed at the protein and, even less, at the mRNA level. Pertaining to these data, a recent study documented maximal vasodilation (>20% compared with control) in perfused rat afferent juxtamedullary arterioles at CO levels in the superfusate of 1.0 µmol/liter (34), which exactly corresponded to the increased CO levels we observed in HUVEC and HAUEC after appropriate E2 stimulation. Despite the fact that HUVEC/HAUEC and perfused rat arterioles are noncomparable systems, it is intriguing to speculate that ovulatory E2 levels might affect vascular tone by CO concentrations previously shown to maximally reducing vascular tone.
In contrast to causing a peak CO release after 4 h of treatment, a 24-h treatment course with ovulatory E2 concentrations no longer increased cellular CO release. As the CO detection system involves measurement of COHb, in contrast to our finding, one might expect COHb levels to accumulate in the medium with time after cell stimulation. By contrast, we could not find such an accumulation of COHb, which is consistent with a previous report demonstrating a 7-fold increase in vascular smooth muscle cell-derived COHb in coculture with HUVEC after 12-h exposure to hypoxia compared with only a 2-fold increase in COHb after 48-h hypoxia (35). Albeit not further discussed in the latter report, we speculate that the lack of COHb accumulation in medium over time observed here and by others (35) most likely reflects dissociation of CO from COHb (36, 37).
We finally were interested in whether CO release could also be attributable to HO1 protein, but, as expected, failed to detect any HO1 protein in HUVECs, which is consistent with recent data obtained from rats showing that HO1 expression was present in the medial, but not endothelial, layer of carotid arteries (38).
There is strong evidence to support the emerging paradigm that CO, like NO, elicits vasodilation (20, 39, 40, 41). However, CO was reported to be less than 1/1000th as potent as NO as a relaxant (20), raising the question of whether vascular CO release by E2 could be of biological significance in terms of effective vasodilation. Recently, chromium mesoporphyrin (CrMP), a selective HO inhibitor, has been shown to increase the myogenic tone of the small muscular branch of rat femoral arteries, but not of large arterial vessels such as the aorta or the femoral artery, in organ bath experiments (21), an effect that could be blunted by coadministration of CO. These researchers further demonstrated that intravascular pressure is required to elicit vasoconstriction by CrMP, that stepwise increases in vascular pressure amplified the constrictor responses of CrMP, and that unpressurized muscle arterioles could not be contracted by CrMP. From these data the researchers suggested that production of CO by vascular HO subserves a vasodilatory mechanism that contributes to the regulation of basal tone in resistance vessels. However, from those data and from the data obtained by Thorup et al. (34) demonstrating the most pronounced vasodilation in renal afferent arterioles at 1.0 µM CO, the biological significance of CO in regulating vascular tone under normal circumstances remained unclear. We have demonstrated here that E2 at normal ovulatory levels can exert endothelial CO release at a concentration comparable to that previously reported to elicit the most pronounced vasodilation.
We further observed high concentrations of E2 had a lesser or no effect on CO and HO2 levels compared with ovulatory E2 concentrations. This inhibitory effect on further CO release and HO2 levels by high doses of E2 might reflect the ability of E2 to maximally induce vasodilation even at higher concentrations shown to markedly stimulate NO release (42), thereby preventing further endothelial CO production, previously shown to reduce arteriolar diameter (34).
Notably, however, the mechanism of how exposure to high levels of E2 prevents further CO release remained to be defined. As high levels of E2 are a potent inducer of endothelial NO release (42), a possible explanation might be the existence of a compensatory interrelationship between the eNOS and HO2 systems in endothelial cells as previously described (43). Using cultured rat endothelial cells, the latter researchers demonstrated that eNOS mRNA was up-regulated in the presence of the heme oxygenase inhibitor zinc protoporphyrin IX, and HO2 mRNA was up-regulated in the presence of a NOS inhibitor. To test whether NOS inhibition in the cells we studied is capable of affecting endothelial CO release, we exposed HAUEC to increasing concentrations of the NOS inhibitor L-NAME in both the absence and presence of E2. Increasing the amount of L-NAME stimulated both basal and E2-mediated CO release. This effect was significant at a high E2 level, but was less pronounced at an ovulatory E2 concentration, suggesting that marked NO release after high dose E2 administration (42) serves to limit the availability of CO. Such an NO-dependent decrease in CO levels might be required to prevent the reduction in arteriolar diameter that was previously shown to result from unopposed release of high amounts of CO (34). However, a possible limitation of our finding is that we did not measure cell culture supernatant NOx levels, e.g. using the Griess detection method, because the entire supernatant in each instance had to be used for CO detection assay. Due to this limitation, we sought to use L-NAME at a maximal concentration 10 times that previously shown to avoid interactions between the NO and CO systems (44).
Another explanation for how E2 prevents from further CO release could be that high levels of E2 inhibit HO2 activity and thereby CO production via a nonreceptor-dependent system, as was suggested for eNOS activity and NOx levels in HUVEC and bovine aortic endothelial cells (4), but no strong evidence for this can be provided here.
Our data showing E2 to be effective in increasing endothelial CO levels are supported by subsequent experiments, demonstrating an increase in HAUEC cGMP levels after an ovulatory E2 dose, thus providing evidence for a functional relevant heme oxygenase-CO-cGMP system in human uterine artery endothelium. However, it is not apparent at present whether ovulatory E2 levels effectively reduce vascular myogenic tone by an endothelial heme oxygenase-CO-cGMP system. Hence, careful and detailed in vivo studies using, for example, ovariectomized animals with E2 replacement should therefore serve to corroborate our data.
In conclusion, our study shows a previously unknown, perhaps physiological function for estrogen in terms of its vasoprotective properties. Our preliminary data indicate that estrogen at a concentration corresponding to the ovulatory phase of the menstrual cycle can serve as a CO-inducing agonist in human endothelial cells. This might be of importance when considering treatment with selective ER modulators such as TAM, which was shown here to blunt the effects of estrogen on endothelial CO release, in the case of underlying vascular disease.
Acknowledgments
We thank Barbara Widmar for her excellent technical assistance.
Footnotes
This work was supported by the Jubiläumsfonds der Österreichischen Nationalbank (Grant 8243). Presented in part at the 47th Meeting of the Society for Gynecologic Investigation, Chicago, Illinois, March 2000.
Abbreviations: CO, Carbon monoxide; CrMP, chromium mesoporphyrin; E2, 17ß-estradiol; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, heme oxygenase; HAUEC, human uterine artery endothelial cells; HUVEC, human umbilical vein endothelial cell; L-NAME, NG-nitro-L-arginine-methyl-ester; NO, nitric oxide; TAM, tamoxifen.
Received July 7, 2000.
Accepted April 7, 2001.
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