Aging-related changes in vascular stiffening and permeability are associated with cardiovascular disease. We examined the interaction of estradiol on the aging process in vascular tissue from rats by assessing the changes in endothelial layer permeability, arterial compliance, and glycoxidative damage levels. We isolated carotid arteries from ovariectomized (OVX) rats that underwent 1 yr of estrogen treatment with subcutaneous pellets and a subsequent 1 mo of cessation of treatment. Endothelial layer permeability and arterial compliance were determined using quantitative fluorescence microscopy. Endothelial layer permeability was reduced with estradiol treatment (estrogen groups, 2.58 ± 0.21 ng dextran · min−1 · cm−2 vs. nonestrogen groups, 4.01 ± 0.30 ng dextran · min−1 · cm−2;P < 0.05). Additionally, arteries from animals treated with estradiol had an increased compliance index (estrogen groups, 82.9 ± 3.8 mm2 · Torr vs. nonestrogen groups, 69.3 ± 3.2 mm2 · Torr; P < 0.05). Estradiol treatment also reduced levels of pentosidine, which is a specific marker of glycoxidative damage (estrogen groups, 0.11 ± 0.03 pmol pentosidine/nmol collagen vs. nonestrogen groups, 0.20 ± 0.03 pmol pentosidine/nmol collagen; P < 0.05). These results indicate that estradiol has multiple chronic vasculoprotective effects on the artery wall to maintain normal vascular wall function.
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.
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.
Pellets containing ovarian sex hormones (90-day release pellets; Innovative Research) were implanted in OVX rats at age 10 wk (4 wk after ovariectomy) as we have described previously (31). OVX animals were separated into five groups, with each group receiving one of these treatments: 17β-estradiol (2.5 mg), progesterone (200 mg), estradiol (2.5 mg) + progesterone (200 mg), no implant (control-OVX), or no hormone (placebo). This procedure was repeated every 3 mo for a total of 4 implants (12 mo of treatment). Experiments were performed 1 mo after cessation of hormone therapy. This approach allowed us to study the chronic effects of estrogen without the complications of the acute effects. Plasma hormone levels taken at the time of the experiments confirmed that all animals had similar low levels of estrogen (12 ± 0.95 pg/ml) at the time that the perfusion experiment was performed.
Pellets were designed to produce estradiol levels in the range of 40–70 pg/ml with the 2.5-mg estradiol pellet and progesterone levels of 15–30 ng/ml with the 200-mg progesterone pellet. To confirm stable hormone release, a few animals (n = 4) were killed early. At 50 days postimplant, estrogen-implanted rats had plasma estradiol levels of 48 ± 2 pg/ml, which dropped to 12 ± 2 pg/ml in animals killed at 3 mo postimplant (at the endpoint of pellet discharge). Additionally, prior use of hormone pellets in a similar group of implanted rats has confirmed stable hormone release for up to 3 mo after pellet administration (30).
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 (If). 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 If involves finding the intersection of tangents that are drawn to approximate these two processes. To determine the accumulation rate, we divide If 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 attime 0 (If0), 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).
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).
Each vessel was preconditioned using three inflation-deflation cycles with pressures from 0 to 150 mmHg to abolish vascular tone and straighten connective tissue fibers (1, 11, 17). Thereafter, each compliance trial lasted 4–5 min, and two trials were performed for each vessel. During inflation, intravascular pressure was increased from 0 to 150 mmHg at a rate of ∼33 mmHg/min. We utilized fluorescence microscopy in addition to video imaging to record vessel-dimension changes because we could achieve greater sensitivity with fluorescence measurements and more data points (which are necessary in performing an accurate nonlinear regression. Video imaging was required to convert our fluorescence measurements to absolute vessel dimensions.
Vessel diameter was measured at 7.5-mmHg hydrostatic pressure increments using direct video imaging of the artery. Because vessel-lumen volume is proportional to vessel fluorescence, we converted the fluorescence measurements detected by the photometer (in mV) into vessel dimension units (in mm). By using both the photometer and the Super VHS video recorder, vessel-dimension changes were obtained at a sampling rate of 1 sample every 2 s. The fit between the recorded diameter changes (from video) and the predicted diameter changes (from fluorescence) consistently produced a coefficient of determination (r 2) > 0.95.
Experiments were performed to investigate the contribution of vascular tone to vessel compliance and distensibility. Carotid arteries were bathed in a solution containing sodium nitrite, and sodium nitrite (10 mM) was added to the superfusate bath between trials to remove all intrinsic vascular tone. All paired trials with and without sodium nitrite yielded similar compliance curves. Additionally, compliance and distensibility measurements done at the beginning and end of the perfusion experiments (2 h) yielded identical compliance and distensibility curves. If vascular tone were contributing to these measurements, one would expect initial measurements of compliance to yield different results than measurements obtained hours later.
Compliance curves were generated by plotting vessel cross-sectional area (S, calculated from diameter) versus intravascular hydrostatic pressure (P; see Fig. 2). With the use of nonlinear regression techniques, these data were fit to the arctangent model of Langewouters and colleagues (11) where α, β, and γ are the parameters that describe the curve. A compliance index (CI) was calculated for each vessel as the area under the compliance curve from 20 to 100 mmHg; this pressure range corresponds to the range of pressures at which these vessels displayed maximal compliance. This calculation involved integrating the Langewouters function using Mathcad 8.0 software (Mathsoft). From this function, specific compliance (C = ΔS/ΔP) can be calculated by the first derivative of the Langewouters function, which yields Vessel distensibility curves were generated by plotting distensibility (calculated as specific compliance ÷ vessel diameter) versus intravascular pressure. A distensibility index (DI) normalizes vascular compliance to vessel size and was calculated (using Mathcad) by integrating the area under the distensibility curve from 20 to 100 mmHg. When vessels under comparison are of different sizes, it is only meaningful to compare DI values to determine vascular stiffening.
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 containingn-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).
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).
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 · min−1 · cm−2 (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 · min−1 · cm−2) 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.3 A). Arteries from animals treated with estradiol had on average a 36% reduction in dextran accumulation (estrogen groups, 2.58 ± 0.21 ng dextran · min−1 ·cm−2 vs. nonestrogen groups, 4.01 ± 0.30 ng dextran · min−1 · cm−2;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.
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. 3 B). 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.
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). Figure5 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. 5 B), artery from an estradiol + progesterone-treated animal without the standard (Fig.5 C; from the same sample as used in Fig. 5 B), and arterial tissue from an OVX animal (Fig. 5 D). 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.
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.
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. 3 A). 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. 3 Band 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.
The authors thank Rebecca Dudovitz for patient and careful work in the laboratory.
This project was supported by the Tobacco-Related Disease Research Program of the University of California (7RT-0070), National Heart, Lung and Blood Institute Grant RO1 HL-55667, and the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research.
Address for reprint requests and other correspondence: J. C. Rutledge, TB 172, 1 Shields Dr., Univ. of California, Davis, CA 95616 (E-mail:).
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.
- Copyright © 2001 the American Physiological Society