INTRODUCTION

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OBSERVATIONAL STUDIES INDICATE that hormone replacement therapy (in particular, estrogen supplementation) is atheroprotective in postmenopausal women (2, 5, 15, 25). Confounding the study of female sex hormones in human health and disease are the diverse actions of these hormones, which include genomic and nongenomic effects on both vascular and nonvascular tissue (7, 14). Although considerable basic research has documented the vasculoprotective actions of estrogens, the precise mechanisms for these effects have been difficult to elucidate in in vivo systems.

The link between enhanced arterial permeability and atherosclerosis has been previously studied (16). There have been conflicting reports as to whether estrogens are capable of modulating arterial permeability (22, 27-29). On the basis of our previous studies (31) where we showed that treatment of lipoproteins and/or the arterial wall with estradiol reduced endothelial layer permeability, we asked the question whether estrogen treatment would have a beneficial effect on arterial wall permeability even after cessation of hormone therapy.

The association between arterial stiffening and atherosclerosis is well known (12, 26). Some authors believe that arterial stiffening as a result of aging or disease can be attributed to the accumulation of nonenzymatic glycoxidation products or advanced glycation end products (AGEs) within the vascular wall (24). Current research indicates that AGE formation occurs with aging, diabetes mellitus, atherosclerosis, and hypertension (3, 10, 18, 20, 26). Hence, glycoxidative stress could accelerate AGE formation in the vascular wall thereby causing structural and mechanical changes to the wall (24, 34). This transduction of glycoxidative stress to arterial stiffening remains incompletely understood. Furthermore, in human studies, estrogen supplementation has been reported to maintain or improve arterial compliance (6, 19). Thus a major goal of this project was to examine the mechanisms by which estrogens may attenuate arterial stiffening.

The present studies demonstrate that chronic estrogen treatment reduces arterial permeability and improves arterial compliance in rats even after cessation of the treatment. Furthermore, in the same arteries, estrogen treatment reduced arterial glycoxidative damage. Combined with our previous study showing a beneficial effect of estrogen treatment on vascular permeability during hormone therapy, we now show that the beneficial effects of estrogen persist after withdrawal of estrogen treatment. Thus our studies demonstrate new, long-lived, and potentially atheroprotective mechanisms of estrogen actions on the vascular wall.

	METHODS

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Chemicals and materials. Krebs-Henseleit buffer consisted of (in mM) 116 NaCl, 5 KCl, 2.4 CaCl₂ · H₂O, 1.2 MgCl₂, 1.2 NH₂PO₄, and 11 glucose. Bovine serum albumin (1% by weight in perfusate) and fluorescently labeled dextran (mol wt 4,400, estimated Stokes diameter 1.4 nm, 42 µg/ml in perfusate) were obtained from Sigma Chemical (St. Louis, MO). Dextran (mol wt 4,400) was labeled with tetramethylrhodamine (TRITC) isothiocyanate (excitation maximum, 494 nm; emission maximum, 518 nm).

Estradiol and progesterone levels. At the time of the experiment, blood was collected from each animal's abdominal vena cava by using a 23-gauge needle and a heparinized syringe. Blood was transferred to sterile Vacutainers and centrifuged at 2,800 rpm for 10 min. Plasma samples were separated from blood cells and kept at 20°C until hormone analysis was performed. Plasma samples were sent to the University of California at Davis Endocrinology Laboratory for radioimmunoassay analysis of estradiol levels and ELISA of progesterone levels.

Animal care and hormone supplementation. Twenty-six ovariectomized (OVX) Sprague-Dawley [Crl:CD(SD)BR strain] rats (age 6 wk) were obtained from Charles River Laboratories. Animals received rat chow and water ad libitum and were kept on a 12:12-h light-dark cycle. All protocols and animal care were approved by the Animal Use Committee at the University of California at Davis.

Measurement of endothelial layer permeability. Carotid arteries from the rats were dissected, cannulated, removed, and placed in a microscope viewing chamber. After the vessel was positioned under the microscope, the rate of TRITC-dextran (mol wt 4,400) accumulation was used to quantitate endothelial layer permeability as previously described (31). Briefly, each trial consisted of 10 min of dextran perfusion and subsequent 10 min of washout. Trials were performed in triplicate to measure the dextran accumulation rate for each vessel. Both carotid arteries were examined in each animal. The measurement of dextran accumulation was performed during the washout phase. Figure 1 illustrates the method employed for estimating dextran accumulation via the measurement of fluorescence intensity (I_f). This estimation involves analyzing washout data as two distinct processes: a rapid washout of the lumen filled with the fluorescent solution and a subsequent, slower, vessel-wall washout. Calculation of accumulation via I_f involves finding the intersection of tangents that are drawn to approximate these two processes. To determine the accumulation rate, we divide I_f by the time of the dye perfusion (10 min). Finally, we use the appropriate conversion factor to convert millivolts per minute to nanograms per minute per centimeter squared. This conversion factor comes from four measurements: 1) the surface area; 2) the lumen volume of the vessel in the photometric window; 3) the maximum fluorescence intensity at time 0 (I_f0), which occurs at the beginning of dye perfusion; and 4) the concentration of dextran. Throughout the perfusion experiment, the vessel was perfused at a rate of 7 ml/min at 37°C and pH 7.4. Distal resistance was adjusted to maintain 100 mmHg of hydrostatic pressure within the vessel at all times. We used dextran because it is a nonlipid reference molecule, and in previous studies we found that it does not specifically bind to the artery wall (8, 31, 32).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Comparison of dextran accumulation in arteries from ovariectomized (OVX) rats. Carotid arteries from OVX rats treated with placebo (control) or estradiol-containing pellets were perfused for 10 min with a buffer solution containing 42 ng/ml tetramethyl rhodamine isothiocyanate (TRITC)-dextran. Arteries were then perfused with nonfluorescent buffer solution that washed out the fluorescent dextran solution from the artery lumen. Fluorescence recordings during washout of TRITC-dextran are shown; these recordings are from two separate experiments and are superimposed to illustrate the differences in dextran accumulation and efflux between both vessels. Perfusion of an artery from the estradiol-treated group is compared with an artery from a rat that was not treated with estradiol. Baseline represents the amount of fluorescence before the beginning of each trial. Both vessels started at similar baseline fluorescence values (~1.3 mV). Intensity measurements of fluorescent dextran accumulation (If) indicate the amount of dextran that accumulated in the artery wall during 10 min of perfusion with the TRITC-dextran solution. Note the decrease in If that was seen with estradiol treatment.

Compliance and distensibility measurements. After the dextran perfusions were performed, static compliance was measured using quantitative fluorescence microscopy and real-time video imaging in the same apparatus as was used for measurement of endothelial layer permeability. The fluorescent TRITC-dextran solution was infused into the proximal cannula of the vessel using an infusion pump. The cannula from the distal end of the vessel was connected to a manometer during the compliance tests to record pressure changes. During compliance tests, the distal end of the tubing was closed. In this way, the infusion pump increased hydrostatic pressure by infusing the TRITC-dextran solution into the vessel at a constant rate. As described in the next paragraphs, the changes in artery size were recorded at increasing hydrostatic pressures by noting changes in fluorescence and artery size (which was observed via video imaging).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Measurements of carotid artery compliance from an OVX rat treated with 2.5 mg of estradiol + 200 mg of progesterone and from an OVX rat treated with a placebo pellet. Note the greater rise in the compliance curve in the artery from the rat treated with estradiol + progesterone, which indicates increased vascular compliance.

Tissue assays. Both right and left carotid arteries from the perfusion studies were washed with PBS buffer and stored at 80°C until use. Both carotids were pooled together for analysis of pentosidine and hydroxyproline contents after the samples were hydrolyzed in 6 N HCl for 24 h at 100°C, methanol-extracted, evaporated under N₂ gas, and finally resuspended in double-distilled water. Arterial pentosidine was measured by high-performance liquid chromatography as we have described previously (30). Briefly, pentosidine was eluted off a C-18 reverse-phase column ioscratically at a flow rate of 1 ml/min using a 15.5% solution of acetonitrile containing n-heptafluorobutyric acid as the counterion. Autofluorescence detection of sample pentosidine was performed at an excitation wavelength of 330 nm and an emission wavelength of 380 nm by using a Hitachi model D fluorometer equipped with a Xe bulb. Pentosidine standard was used to confirm pentosidine peaks in samples, which were eluted off at ~22-24 min in all samples. Normalization of samples was performed by injecting a constant amount (15 µg) of hydroxyproline (collagen) for each sample. Pentosidine levels are expressed relative to collagen contents. Arterial collagen (hydroxyproline) levels were determined by colorimetric analysis of sample hydrolysates using the Woessner assay (30).

Statistical analysis. One-factor ANOVA was used to determine the presence of any differences that existed between our four groups. The Bonferroni multiple-comparison test was used to report the magnitude of the differences found in animals receiving estradiol and those not receiving estradiol. The animals receiving placebo implants and the control OVX were placed in the same groups in accordance with their similar responses to treatment (i.e., estrogen and progesterone levels). Tests of significance were applied at the 5% level. The statistical analyses were aided by the use of SigmaStat 2.0 software (Jandel Scientific Software).

	RESULTS

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Effects of estradiol treatment on endothelial layer permeability. Using quantitative fluorescence microscopy, we measured the rate of TRITC-dextran accumulation in individually perfused arteries. OVX animals treated with estradiol (both the estradiol-only group and the estradiol + progesterone group) had a dextran accumulation rate that was 0.32-2.51 ng · min¹ · cm² (or 6-47%) less relative to the groups not receiving estradiol [the OVX, placebo (OVX treated with placebo), and progesterone (OVX treated with progesterone) groups]; the confidence interval was 95% with two-factor ANOVA and the Bonferroni post hoc test. The rates of dextran accumulation in each group were (in ng · min¹ · cm²) estradiol, 4.17 ± 0.49; estrogen + progesterone, 3.46 ± 0.33; progesterone, 4.97 ± 0.41; control (OVX), 5.20 ± 0.69; placebo, 5.78 ± 0.95 (P < 0.05; see Fig. 3A). Arteries from animals treated with estradiol had on average a 36% reduction in dextran accumulation (estrogen groups, 2.58 ± 0.21 ng dextran · min¹ ·cm² vs. nonestrogen groups, 4.01 ± 0.30 ng dextran · min¹ · cm²; P < 0.05). These data are consistent with our previous results using dextran with mol wt 76,000 (31). No significant differences were noted between the control and placebo-treated groups in any of the analyses; therefore they are considered as one group.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A: effects of estradiol treatment on endothelial layer permeability. Rate of dextran accumulation was measured in each carotid artery from each treatment group. Estradiol-supplemented rats had 6-47% reduced endothelial layer permeability relative to animals without estrogen supplementation (*P < 0.05). B: effects of estradiol treatment on vascular stiffening. Compliance index of each artery was calculated as the area under the compliance curve from 20 to 100 mmHg. Estradiol treatment enhanced vascular compliance by 5-40% (*P < 0.05). E2, estradiol; P4, progesterone.

Arterial compliance. After the permeability measurements, the CI was determined in each artery from each of the treatment groups. Hormone status had no effect on vessel size. Arteries from animals that received estradiol had a vascular compliance that was 5-40% greater (confidence interval of 95%; two-factor ANOVA and Bonferroni post hoc test). The group compliance indices were (in mm² · Torr) estradiol, 77.6 ± 3.0; estrogen + progesterone, 90.8 ± 3.1; progesterone, 67.9 ± 3.3; and control/placebo (no hormone), 69.7 ± 4.1 (P < 0.05; see Fig. 3B). On average, there was a 20% increase in vascular compliance with estrogen supplementation (estrogen groups, 82.9 ± 3.8 mm² · Torr vs. nonestrogen groups, 69.3 ± 3.2 mm² · Torr; P < 0.05). An analysis of vascular distensibility, which normalizes vascular compliance to vessel size, showed the same trend with arteries from estradiol-treated animals having vascular distensibility that was 34% greater (P < 0.05). The group DI values were (in mm² · Torr): estradiol, 0.426 ± 0.012; estrogen + progesterone, 0.519 ± 0.060; progesterone, 0.383 ± 0.041; and control/placebo (no hormone), 0.335 ± 0.037 (P < 0.05; see Fig. 4). Thus these experiments showed that estradiol treatment improved both arterial compliance and distensibility.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of estradiol treatment on arterial distensibility. Distensibility index of each artery was calculated as the area under the distensibility curve from 20 to 100 mmHg. Animals receiving estrogen treatment had on average 34% greater distensibility index (*P < 0.05).

Glycoxidative damage. We related our functional studies of compliance with biochemical changes in the artery wall by measuring pentosidine (which is a highly specific marker of glycoxidative damage and crosslinking) in pooled arteries from each of the treatment groups. When all of the vessels treated with estradiol (n = 11) were compared with all of the non-estradiol-treated vessels (n = 10), there was an ~50% reduction in pentosidine (0.11 ± 0.03 pmol pentosidine/nmol collagen vs. 0.20 ± 0.03 pmol pentosidine/nmol collagen; P < 0.05). Figure 5 illustrates representative chromatographs from our pentosidine standard and from these carotid artery samples: standard plus a carotid artery from an estradiol + progesterone-treated animal (Fig. 5B), artery from an estradiol + progesterone-treated animal without the standard (Fig. 5C; from the same sample as used in Fig. 5B), and arterial tissue from an OVX animal (Fig. 5D). Our previous studies with rats (30) showed a similar reduction in pentosidine from the iliac arteries of rats treated with estradiol relative to control and placebo. Furthermore, the collagen content of estrogen-treated arteries did not change significantly from nonestrogen-treated arteries in that study.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of estradiol on vascular glycoxidative levels. Pentosidine, a specific advanced glycoxidative end product, was measured in each artery from each treatment group. These are representative chromatographs from carotid artery hydrolysates. Shaded peaks represent pentosidine levels in each sample; numbers above each are times of peak elution. A: pentosidine standard. B: pentosidine standard + an arterial tissue sample from an animal treated with estradiol and progesterone. C: arterial tissue sample used in part B (without the standard). D: arterial tissue from an OVX animal. Note the increased size of the pentosidine peak from the animal not receiving estrogen. Across all animals and groups, estradiol treatment was associated with reduced arterial pentosidine.

Effect of hormone treatment on weight and sex hormones. Body weight and fasting plasma concentrations of estradiol and progesterone were measured in each animal from each treatment group on the day of the experiment (see Table 1). In these animals, weight was not significantly different among the groups, although when we consider all of the animals receiving estrogen, we see that there is a significant difference in body weight between estradiol and non-estradiol-treated groups (estradiol, 413 ± 21 g vs. nonestradiol, 486 ± 22 g; P < 0.05). One month after the pellets were expected to have stopped releasing hormone, plasma estradiol concentrations were low (12 ± 0.95 pg/ml) and virtually identical in all treatment groups. Plasma progesterone was significantly different in the progesterone and estradiol + progesterone groups relative to the groups that did not receive progesterone implants (49.1 ± 10.0 vs. 7.1 ± 0.9 ng/ml; P < 0.05). This may reflect the presence of residual progesterone or one of its metabolites detected in the ELISA assay. However, as noted above, this residual progesterone apparently had no beneficial effect on endothelial layer permeability or compliance.

	DISCUSSION

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In this study we showed that chronic estrogen supplementation in OVX rats: 1) reduced endothelial layer permeability, 2) attenuated arterial stiffening, and 3) reduced the AGE pentosidine. These results indicate that estrogens have multiple potential beneficial effects on the artery wall even after cessation of estradiol treatment. Furthermore, our study indicates mechanisms by which estrogens reduce arterial permeability and stiffening.

For this report, we utilized a new method for sequentially measuring endothelial layer permeability, arterial compliance, and biochemical parameters in the same arteries. Using this system, we are able to more precisely investigate the mechanisms and effects of vascular stiffening. The advantage of our system is that functional (e.g., vascular compliance and permeability) and biochemical (e.g., pentosidine) measurements can be made on the same tissue sample. Additionally, our perfusion apparatus enables close monitoring of important physiological parameters such as flow, temperature, pH, and hydrostatic pressure during the functional tests.

In light of the conflicting reports of the effects of estrogen on vascular wall permeability, we have now repeatedly demonstrated a beneficial effect of estrogen on permeability in both acute (8, 32) and chronic settings (31). This paper builds on our previous work (31), which showed a reduction of permeability with estradiol supplementation in rats. In our previous study, experiments were conducted during estradiol supplementation. Because estradiol was still present in the vascular tissue at the time of the experiment, we could not separate the direct antioxidative effects of estrogen from the chronic or genomic effects. In the present study, estradiol levels in all treatment groups were the same at the time of the experiments (see Table 1). Despite this, a reduction in vascular permeability was maintained in the animals that received estradiol (see Fig. 3A). Hence, it appears that estradiol is acting to maintain vascular function by effecting processes that are long lived. It is unknown whether the actions of estrogen are to decrease or preserve permeability, or whether the lack of estrogen increases endothelial permeability.

Arterial stiffening has been attributed to a number of pathophysiological processes (4, 24, 26). Potential etiologies include increased arterial collagen, alteration in endothelium-derived relaxation, and nonenzymatic glycation of elastin or collagen (17, 35). Most recent studies point to collagen crosslinking as the primary etiological factor seen in diabetic and atherosclerotic arterial stiffening (24, 35). Oxidant or glycoxidative stress may stimulate collagen crosslinking by the incorporation of glucose-derived crosslinks (such as pentosidine) between collagen fibers. This crosslinking then reduces vascular distensibility to arterial pulsatile forces.

Our results support the hypothesis that arterial stiffening is generated by nonenzymatically derived collagen crosslinks. Consistent with this hypothesis and with our previous study (30), we detected increased pentosidine levels in stiffened vessels from OVX rats compared with estradiol-treated rats. Our physiological measurements of vascular stiffening showed improved compliance and distensibility in estrogen-treated animals (see Figs. 3B and 4). These data suggest that estrogen prevents vascular stiffening by attenuating glycoxidative damage and collagen crosslinking.

It is interesting to note that the combination of both progesterone and estradiol appears to provide more vascular protection with regard to permeability and compliance (see Figs. 3 and 4). When coupled with estradiol treatment, this beneficial effect of progesterone treatment was more pronounced in compliance, where the differences between the combination group and the estradiol group approached significance (P = 0.08). Without estradiol, this protective effect of progesterone is not seen, which possibly suggests a synergistic relationship between progesterone and estradiol.

The association between vascular stiffening and increased vascular permeability has not been reported before. Cell-culture studies (9) have shown a causal relationship between cultured endothelial monolayer permeability and altered matrix components. Other studies have demonstrated that soluble AGEs increase vascular permeability via the AGE receptor (21, 26, 33). Further studies are needed to better understand the relationship between endothelial layer permeability and vascular stiffening and determine whether there may be a causal or associative role between the two.

The mechanism(s) by which estrogens modulate these vascular changes are complex. We and others have shown that at physiological and supraphysiological concentrations, estrogens have direct antioxidant effects (8, 13, 14, 23, 31, 32). Further, our previous work (30) showed that estrogen reduced vascular levels of hydroperoxides. Thus by scavenging reactive oxygen species, estrogens may prevent oxidative and glycoxidative damage and subsequent collagen crosslinking and vascular stiffening. These effects could produce long-term changes in vascular function that are maintained after cessation of estrogen supplementation.