Absence of Endothelial ERα Results in Arterial Remodeling and Decreased Stiffness in Western Diet–Fed Male Mice

Abstract

The role of estrogen receptor-α (ERα) signaling in the vasculature of females has been described under different experimental conditions and our group recently reported that lack of endothelial cell (EC) ERα in female mice fed a Western diet (WD) results in amelioration of vascular stiffness. Conversely, the role of ERα in the male vasculature in this setting has not been explored. In conditions of overnutrition and insulin resistance, augmented arterial stiffness, endothelial dysfunction, and arterial remodeling contribute to the development of cardiovascular disease. Here, we used a rodent model of decreased ERα expression in ECs [endothelial cell estrogen receptor-α knockout (EC-ERαKO)] to test the hypothesis that, similar to our findings in females, loss of ERα signaling in the endothelium of insulin-resistant males would result in decreased arterial stiffness. EC-ERαKO male mice and same-sex littermates were fed a WD (high in fructose and fat) for 20 weeks and then assessed for vascular function and stiffness. EC-ERαKO mice were heavier than littermates but exhibited decreased vascular stiffness without differences in endothelial-dependent vasodilatory responses. Mesenteric arteries from EC-ERαKO mice had significantly increased diameters, wall cross-sectional areas, and mean wall thicknesses, indicative of outward hypertrophic remodeling. This remodeling paralleled an increased vessel wall content of collagen and elastin, inhibition of matrix metalloproteinase activation and a decrease of the incremental modulus of elasticity. In addition, internal elastic lamina fenestrae were more abundant in the EC-ERαKO mice. In conclusion, loss of endothelial ERα reduces vascular stiffness in male mice fed a WD with an associated outward hypertrophic remodeling of resistance arteries.

Loss of endothelial estrogen receptor-α reduces vascular stiffness in obese male mice fed with an associated outward hypertrophic remodeling of resistance arteries.

Consumption of diets high in fat and fructose [Western diet (WD)] is associated with the alarming prevalence of insulin resistance, obesity, and type 2 diabetes (1–3). Importantly, cardiovascular disease (CVD) remains a leading cause of death in the diabetic and obese population (4). Endothelial dysfunction, vascular remodeling, and augmented arterial stiffness are among the vascular defects that lead to CVD in conditions of insulin resistance (5–7). We have previously demonstrated that WD feeding results in endothelial dysfunction, vascular remodeling, and augmented stiffness in large arteries of both male and female mice (8, 9). In addition, we have shown that WD feeding causes stiffening of resistance arteries and vascular remodeling, characterized by increased elastin content and decreased fenestration of their internal elastic lamina (IEL) (10). As estrogenic action in the vasculature has been classically described as protective against CVD (11), we recently began to explore the role of endothelial estrogen signaling in the genesis of CVD caused by obesity and insulin resistance. Vascular actions of estrogens are mediated via both intracellular and membrane-bound receptors (11, 12). Among these, stimulation of estrogen receptor-α (ERα) is known to positively impact, under normal conditions, endothelial nitric oxide (NO) bioavailability (11) and vascular remodeling (13). We recently examined the effect of endothelial ERα deficiency on vascular stiffness in obese insulin resistant female mice (14). Contrary to our hypothesis, we found that female mice lacking endothelial cell (EC) ERα were protected against WD-induced vascular stiffness despite worsening glucose homeostasis (14). However, whether similar findings occur in males remains unknown. Although present both in females and males, the role of ERα in the vasculature of males has not been extensively characterized. Evidence exists that in the nonobese state, there is a positive effect of ERα only on basal production of NO (15), whereas others have reported that lack of estrogen signaling results in impaired endothelial-dependent dilation without impacting vascular stiffness (16, 17). This paucity of data led us to investigate whether EC-specific ablation of ERα would impact endothelial function and vascular remodeling in male mice fed a WD. We hypothesized that, similar to females, loss of ERα signaling in the endothelium of males would result in decreased arterial stiffness in the setting of WD consumption.

Methods

Animals

Animal procedures were performed in accordance with the Animal Use and Care Committee at the University of Missouri–Columbia and National Institutes of Health guidelines. The Animal Use and Care Committee at the University of Missouri–Columbia reviewed and approved the animal protocol followed. We studied only males in the present work, as we previously reported the effect of EC ERα-specific knockout (KO) in females (14). We used the ERα EC-specific KO (EC-ERαKO) mouse model and littermate controls [estrogen receptor-α floxed (ERαFl₂)]. These mice were generated as previously described (14). Briefly, mice with lox P sites encompassing exon 3 of the ERα gene (18) were sequentially crossed with Cad-Cre–positive mice [vascular endothelial (VE)–cadherin promoter driving expression of Cre-recombinase] according to standard procedures to obtain double floxed ERα Cre-positive mice (EC-ERαKO) and double-floxed ERα VE-cadherin Cre-negative mice (ERαFl₂). The VE-cadherin Cre recombinase used in this model has been previously shown to be expressed in adult ECs (19). Beginning at 8 weeks of age, both cohorts of mice were fed a WD consisting of high fat (46%), high carbohydrate as sucrose (17.5%), and high-fructose corn syrup (17.5%) for 20 weeks (Test Diet 58Y1, 5APC Richmond, Indiana). This diet contains casein as a protein source and no phytoestrogens. We have previously shown that this feeding paradigm results in whole-body insulin resistance (20). All cohorts were provided water ad libitum while housed in pairs under a 12-hour/day illumination regimen.

Body weight, fasting glucose, insulin, and estradiol

To assess weight gain, mice were weighed immediately prior to killing (21). In addition, 5-hour fasting serum samples were obtained for insulin and glucose measurements. Hepatic insulin resistance was determined using the homeostatic model assessment of insulin resistance index, which was calculated as fasting insulin (μU /mL) × fasting glucose [mg/dL] ÷ 405 (22). Estradiol was determined by radioimmunoassay at the Vanderbilt University Medical Center Hormone Assay and Analytical Services Core, which is supported by National Institutes of Health Grants DK059637 and DK020593.

Intraperitoneal glucose tolerance test

Intraperitoneal glucose tolerance test (IPGTT) was performed in the cohorts after a 5-hour fast as previously described (14). Briefly, dextrose (1.0 g/kg) was injected intraperitoneally, and the glucose excursion was monitored over a 2-hour time course and compared between treatment groups. Blood samples were analyzed for glucose (AlphaTRACK) at time 0 and 15, 30, 45, 60, and 120 minutes after dextrose injection. The cumulative glycemic excursion was evaluated as the area under the curve (14).

Polymerase chain reaction for genomic DNA

Polymerase chain reaction (PCR) for genomic DNA was performed as previously described (14). The primer sequences used were 5′-GAACCTGATGGACATGTTCAGGGA-3′, Rev:5′ CAGAGTCATCCTTAGCGCCGTAAA-3′ for Cre, and 5′-TTGCCCGATAACAATAACAT-3′, Rev:5′-TGCAGCAGAAGGTATTTGCCTGTTA-3′ for ERα.

Quantitative Real-Time PCR

For quantitative PCR, total RNA was isolated from the primary cultured ECs using TRIzol reagent (Sigma, St. Louis, MO). RNA yield was determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). First-strand complementary DNA synthesis was done using 1 μg total RNA with oligo dT (1 μg), 5× reaction buffer, MgCl2, dNTP mix, RNAse inhibitor, and Improm II reverse transcription as per Improm II reverse transcription kit (Promega, Madison, WI). After the first-strand synthesis, real-time PCR was done using 8 μL complementary DNA, 10 μL SYBR green PCR master mix (Bio-Rad Laboratories, Hercules, CA) and forward and reverse primers (10 pM/μL; Integrated DNA Technologies, San Diego, CA) using a real-time PCR system (CFX96; Bio-Rad Laboratories). The primer sequences used for ERα were forward: 5′-CTGTCGGCTGCGCAAGTGTT-3′; reverse: 5′-CATCTCTCTGACGCTTGTGCT-3′ (NM_001302533.1). The specificity of the primers was analyzed by running a melting curve. The PCR cycling conditions used were 5 minutes at 95°C for initial denaturation, 39 cycles of 30 seconds at 95°C, 30 seconds at 58°C, and 30 seconds at 72°C. Calculations of relative normalized gene expression were done using the Bio-Rad CFX manager software based on the delta cycle threshold (ΔCt) method. The results were normalized against housekeeping gene GAPDH.

Isolation of lung ECs

ECs were isolated from each mouse via two-step magnetic isolation method with modifications (23). Lungs were chopped finely and digested with lung dissociation enzymatic reagent (Lung dissociation kit, 130-095-927, Milteny Biotech, San Diego, CA) for 90 minutes at 37°C. The digested tissue was then dissociated using a 3-mL syringe plunger and filtered through a 30-µm disposable cell strainer (Milteny Biotech, San Diego, CA) and centrifuged at 300 g for 10 minutes at 4°C. To remove CD45 leukocytes that express CD31, CD45 microbeads (CD 45 microbeads, 130-052-301, Milteny Biotech) were added to cell suspension, mixed and incubated for 15 minutes at 4°C and then passed through a magnetic column. ECs present in the eluent were pelleted and incubated with CD31 coated magnetic beads (CD31 microbeads, 130-097-418, Milteny Biotech) for 15 minutes at 4°C and then passed through a magnetic column. ECs bound to CD31 magnetic beads were then recovered and used for primary culture. Cells at passage 1 were used for messenger RNA (mRNA) expression.

Aortic stiffness by in vivo pulse wave velocity

In vivo arterial stiffness was evaluated via pulse wave velocity (PWV) Doppler ultrasound (Indus Mouse Doppler System, Webster, TX) as previously described (24). All procedures were performed on isoflurane-anesthetized mice (1.75% in 100% oxygen stream) (8).

Assessment of arterial vasomotor responses ex vivo

Aorta

A segment of thoracic aorta, trimmed of fat and connective tissue, was sectioned into a 2-mm ring and mounted on a wire myograph chamber (Model 620M, Danish Myo Technology, Aarhus, Denmark) containing physiological salt solution gassed with 95% O₂ and 5% CO₂ at 37°C, as previously described (25, 26). After a 30-minute equilibration period, aortic rings were brought to an optimal tension and then another 30 minutes of equilibration followed. Rings were stimulated with 80 mM KCl to assess vessel viability. Aortic rings were preconstricted with 10⁻⁶ M of phenylephrine and vasomotor reactivity was assessed with cumulative concentration-response curves of acetylcholine [(ACh) 10⁻⁹ to 10⁻⁵ M] and the NO-donor sodium nitroprusside [(SNP) 10⁻⁹ to 10⁻⁵ M]. Relaxation at each concentration was measured and expressed as percent maximum relaxation, where 100% is equivalent to loss of all tension developed in response to phenylephrine.

Mesenteric arteries

Vascular reactivity was evaluated in second-order mesenteric resistance arteries as previously described (27). Briefly, arterial segments viability was tested by exposure to 80 mM KCl. Endothelium-dependent vasodilation was assessed by cumulative exposure to increasing concentrations of ACh (10⁻⁹ to 10⁻⁵ M) in arteries preconstricted with 10⁻⁵ M phenylephrine. In addition, arteries were also exposed to cumulative concentrations of SNP (10⁻⁸ to 10⁻⁴ M) to evaluate endothelium-independent vasodilatory responses in arteries preconstricted with 10⁻⁵ M phenylephrine.

Assessment of mesenteric artery stiffness and structural remodeling

At the end of the reactivity experiments the same set of mesenteric arteries were analyzed under passive conditions to determine their mechanical and structural characteristics. Vessels were washed three times in a calcium-free buffer that contained 2 mM EGTA and 10⁻⁴ M adenosine to ensure passive conditions. Vessels were then exposed to consecutive 2-minute changes in intraluminal pressure from 5 to 120 mm Hg while under passive conditions to determine the elastic properties of the arteries.

During the duration of the experiment, chambers were mounted on inverted microscopes with charge-coupled device cameras. Luminal diameter and wall thicknesses were recorded using a video caliper (Living Systems Instrumentation, Burlington, VT) and a Powerlab data acquisition system (ADInstruments Inc., Colorado Springs, CO).

Confocal/multiphoton microscopy imaging of mesenteric arteries

Confocal microscopy images were obtained as previously described (28). Briefly, vessels were fixed, while pressurized, in 4% paraformaldehyde. Ahead of imaging, vessels were rinsed in phosphate-buffered saline and in 0.1 M Glycine. After cannulation, vessels were flushed with phosphate-buffered saline and permeabilized via incubation in 0.5% Triton X-100, followed by incubation for 1 hour in 0.5 μg/mL 4',6-diamidino-2-phenylindole, 0.2 μM 5 Alexa Fluor 633 Hydrazide (Molecular Probes) and 0.02 μM Alexa Fluor 546 phalloidin (Molecular Probes). Lastly, vessels were imaged using a Leica SP5 confocal/multiphoton microscope with a 63×/1.2 numerical aperture water objective. Alexa Fluor 633 was used to stain elastin. Alexa Fluor 546 phalloidin was used to stain F-actin, and 4',6-diamidino-2-phenylindole was used to stain nuclei. Collagen was imaged via second-harmonic image generation using a multiphoton laser at 850 nm. The number and area of fenestrae in the IEL were quantified using images generated with Alexa 633. All imaging and image analyses were performed as previously described (27–30).

Detection of matrix metalloproteinase activity by in situ zymography

Gelatinolytic activity in isolated arteries was measured via in situ zymography as previously described (30). Briefly, a segment of a mesenteric artery was carefully dissected, cleaned, and cannulated onto a pressure myograph (Living Systems). The artery was then equilibrated for 1 hour under 70 mm Hg intraluminal pressure at 37°C without flow. After equilibration, arteries were transferred to a confocal/multiphoton microscope system and exposed for the rest of the experiment to fluorogenic dye-quenched (DQ) gelatin (10 µg/mL), which, upon digestion by the gelatinases matrix metalloproteinase (MMP)-2 and MMP-9, emits green fluorescent light. DQ gelatin was applied abluminally to all vessels. Five minutes after the application of DQ gelatin, the whole artery was imaged with a Leica SP5 confocal/multiphoton microscope using a 20×/0.7 numerical aperture air objective (31). DQ gelatin was excited with a wavelength of 800 nm and emission detected with nondescanned detectors at a wavelength of 500 to 550 nm. Optical z sections were 2 µm thick. Right after the first image was taken, 17β-estradiol (10 µM) was added intraluminally, and 5 minutes after its application, an additional image was taken. Thereafter, an image was taken every hour for 4 hours using the same parameters as above. Image reconstructions were performed using Imaris software (Bitplane). The fluorescence intensity was calculated for every three-dimensional image of the whole artery (31). All the images acquired had the same volume (1024 × 1024 × 170 pixels). We expressed the changes in fluorescence intensity over time as percent changes in fluorescence intensity from the first image taken after the initial exposure to DQ gelatin (31).

Immunohistochemistry

A segment of thoracic aorta was fixed in 3% paraformaldehyde, dehydrated in ethanol, paraffin embedded, and transversely sectioned in 5-µm slices. Four sections for each mouse were examined. To evaluate remodeling as previously described, sections were stained with Verhoeff-von Gieson (VVG) and Trichrome (8). VVG was quantified as the average percent intensity of staining in five images per aorta. Trichrome staining was used to quantify the thickness of the media. These measurements were obtained at 4× magnification using the ImageJ/Fiji image analysis platform.

Statistical analysis

Results are reported as means ± standard errors (standard error of the mean). Differences in outcomes were determined using primarily independent two-tailed t tests and repeated-measures analysis of variance (Sigma Plot 13.0, Systat Software, Point Richmond, CA). A P ≤ 0.05 was considered statistically significant.

Results

ERα expression is significantly decreased in ECs of EC-ERαKO mice

To determine the effectiveness of our knockout procedure, we measure mRNA signal for ERα in ECs isolated from the animal cohorts used in this investigation. We found that ERα mRNA was significantly decreased in cells isolated from EC-ERαKO mice compared with ERαFl₂ [Fig. 1(a)].

Figure 1.

Decreased expression of ERα in ECs in the setting of WD-feeding results in enhanced endothelial-independent dilation and decreased aortic stiffness. (a) Reduced ERα mRNA in primary ECs cultured from mouse lungs. Vasodilatory responses of isolated aortic rings to (b) the endothelium-dependent dilator, ACh, and (c) the endothelium-independent dilator, SNP. (d) Aortic PWV values obtained before kill after 20 weeks of WD feeding. (e) Aortic media thickness and (f) VVG staining intensity as a measurement of elastic fibers content. Values are mean ± standard error of the mean; n = 6 to 8 for all groups. *P < 0.05.

WD results in greater weight gain in EC-ERαKO mice

Independent of sex, global KO of ERα has been shown to result in obesity and systemic insulin resistance (20). We have also previously shown that in females, endothelial-specific deletion of ERα results in a greater gain of visceral fat, as well as a greater impairment in glucose homeostasis when mice were fed a WD (14). In the present investigation, 20 weeks of WD in males resulted in greater body weight in the EC-ERαKO compared with littermate controls ERαFl₂ (Table 1). We fed males for 20 weeks with WD because our previous investigation showed that a shorter feeding (8 weeks) did not result in increased cardiovascular stiffness in males (20). In vivo glucose homeostasis was evaluated via IPGTT with 1.0 g/kg of dextrose and we found no significant differences between the two cohorts in this parameter (Table 1). However, there was a trend toward increased insulin resistance, examined by the homeostatic model assessment of insulin resistance, in the EC-ERαKO when compared with ERαFl₂ (P = 0.07; Table 1). Plasma estradiol levels were not significantly different between the KO mice and the littermates (Table 1).

Table 1.

Body Weight and Biochemical Parameters

	Body Weight, g	Glucose, mg/dL	Insulin, ng/mL	Homeostatic Model Assessment of Insulin Resistance	Glucose Tolerance Test Area Under the Curve (Arbitrary Units)	Estradiol (pg/mL)
ERαFl2	33.57 ± 0.96	261.38 ± 19.00	1.07 ± 0.26	14.56 ± 3.23	31.01 ± 3.69	27.57 ± 3.14
EC-ERαKO	37.36 ± 1.24a	291.83 ± 12.73	1.91 ± 0.55	29.09 ± 7.51b	25.21 ± 3.12	34.18 ± 3.15

Values are mean ± standard error of the mean, n = 6 to 8.

^aP ≤ 0.05 ERαFl₂ vs EC-ERαKO.

^bP = 0.07.

EC-ERαKO mice exhibit greater endothelial-independent aortic dilation and less aortic stiffness

Our work in females revealed that an absence of ERα in the endothelium results in decreased vascular stiffness when mice were fed a WD for 8 weeks (14). Herein, we evaluated endothelial-dependent and endothelial-independent aortic vasodilatory responses in male mice fed a WD for 20 weeks, as these have been related to the genesis of vascular stiffness (3). Endothelial-dependent dilation was examined with dose-responses to ACh, which were not different between the two cohorts [Fig. 1(b)]. Conversely, EC-ERαKO mice had a greater response to SNP than ERαFl₂ mice [Fig. 1(c)], suggesting that an absence of endothelial ERα leads to enhanced endothelial-independent dilation. Aortic tension developed in response to phenylephrine was similar between the groups (4.361 ± 0.285 mV vs 4.098 ± 0.515 mV, P = 0.665, for ERαFl₂ vs EC-ERαKO, respectively). In addition, we assessed in vivo aortic stiffness via PWV and found less stiffness in EC-ERαKO when compared with ERαFl₂ [Fig. 1(d)].

We then examined changes corresponding to aortic remodeling. There was no significant change in regards to medial thickness between the two cohorts evaluated by trichrome staining [Fig. 1(e)]. In addition, the intensity of VVG staining as a marker of elastic fiber content in the thoracic aorta showed a nonsignificant tendency for a higher elastin content in the KO mice [P = 0.068; Fig. 1(f)].

Absence of endothelial ERα results in outward hypertrophic remodeling and decreased stiffness in resistance vessels

Our group has previously shown that under WD feeding conditions, male mice exhibit increased mesenteric artery stiffness without changes in vasomotor responses compared with control diet fed mice (10). In the current study, we examined the role of endothelial ERα in modulating the vasomotor and mechanical responses of mesenteric arteries in the setting of WD feeding in male mice. There were no significant differences in the vasomotor responses to phenylephrine, Ach, or SNP between the two cohorts [Fig. 2(a–c)]. We then assessed the mechanical properties of these vessels, documenting increased passive internal and external diameter, mean-wall thickness and cross-sectional area in the EC-ERαKO cohort when compared with the ERαFl₂ cohort [Fig. 3(a–d)]. Interestingly, there was a lower modulus of elasticity and incremental modulus of elasticity, and a higher cross-sectional compliance in the EC-ERαKO mice when compared with the ERαFl₂ cohort [Fig. 3(g–i)]. These results indicate that under WD feeding conditions, lack of endothelial ERα results in decreased arterial stiffness despite the presence of an outward hypertrophic remodeling.

Figure 2.

EC-ERαKO does not alter vasomotor responses in resistance vessels under WD feeding conditions. Vasomotor responses of isolated second-order mesenteric arteries to (a) phenylephrine (Phe), (b) endothelial-dependent vasodilator ACh, and (c) endothelial-independent vasodilator SNP. n = 6 to 8 per group.

Figure 3.

EC-ERαKO results in outward hypertrophic remodeling and decreased stiffness of resistance arteries after 20 weeks of WD feeding. (a) Passive internal, (b) external diameter at different intraluminal pressures, (c) mean wall thickness–pressure relationships, (d) vascular wall cross-sectional area (CSA)–pressure relationships, and (e) wall-to-lumen ratio. (f) Strain–stress relationships, (g) Young and (h) incremental modulus of elasticity–pressure curves, and (i) vessel cross-sectional compliance (CSC). n =6 to 8 per group. *P ≤ 0.05 EC-ERαKO vs ERαFl₂.

Effect of loss of endothelial ERα on extracellular matrix content and MMP activity in mesenteric resistance arteries

To determine the contribution of vascular smooth muscle cells (VSMCs) and extracellular membrane components on the mechanical changes seen in mesenteric arteries, we imaged and quantified VSMC nuclei, VSMC F-actin content, elastin, and collagen in vessels from both cohorts [Fig. 4(a–d)]. There were no statistically significant differences in the number of VSMCs or F-actin content between the two cohorts [Fig. 4(e–f)]. Nevertheless, there was a significant increase in the content of elastin and collagen in EC-ERαKO mice [Fig. 4(g) and 4(h)], without changes in the elastin to collagen ratio [Fig 4(i)]. We then examined the impact of loss of EC-ERα in the activation of MMPs in mesenteric arteries. There was a significant decrease in MMP activity induced by estrogen treatment in the EC-ERαKO mice when compared with controls (Fig. 5). Finally, the number of fenestrae and area per fenestra in the IEL were also evaluated. As shown in Fig. 6, EC-ERαKO mice had a significant increase in the number of mesenteric artery fenestrae [Fig. 6(a–c)], whereas the mean area occupied by each fenestra was decreased [Fig. 6(d)]. In addition, there was a trend (P = 0.13) toward a decrease in the modulus of elasticity specific for the [IEL at 70 mm Hg (E/E0); Fig. 6(e)], which suggests vessels tended to be more pliable.

Figure 4.

EC-ERαKO in the setting of WD feeding increases elastin and collagen content in resistance arteries. Representative images of a vessel stained with (a) 4',6-diamidino-2-phenylindole to image nuclei, (b) Alexa Fluor 546 Phalloidin to image F-actin, (c) Alexa Fluor 633 Hydrazide to image elastin (left panel, ERαFl₂; right panel, EC-ERαKO), and (d) subjected to second harmonics to image collagen (left panel, ERαFl₂; right panel, EC-ERαKO). Scale bar = 30 μm. (e) Comparison of number of voxels containing 4',6-diamidino-2-phenylindole fluorescence in the medial layer of mesenteric arteries from ERαFl₂ and EC-ERαKO mice. (f) Comparison of number of voxels containing Alexa Fluor 546 Phalloidin fluorescence in the medial layer of mesenteric arteries from ERαFl₂ and EC-ERαKO mice. (g) Comparison of number of voxels containing Alexa Fluor 633 Hydrazide fluorescence in the wall of mesenteric arteries from ERαFl₂ and EC-ERαKO mice. (h) Comparison of number of voxels containing second harmonic–detected collagen in the wall of mesenteric arteries from ERαFl₂ and EC-ERαKO mice. (i) Comparison of the ratio of the number of voxels containing Alexa Fluor 633 Hydrazide fluorescence over those containing second harmonic–detected collagen in the wall of mesenteric arteries from ERαFl₂ and EC-ERαKO mice. (e–i) Data are expressed as means ± standard error of the mean. n = 6 to 8 per group. *P ≤ 0.05 EC-ERαKO vs ERαFl₂.

Figure 5.

(a and b) Loss of ERα signaling in the endothelium inhibits estradiol-induced MMP activation in isolated, cannulated, and pressurized mesenteric resistance arteries. Arteries from mice lacking or not lacking ERα on their endothelium were incubated for 4 hours with 17β-estradiol (10 µM) in the presence of DQ gelatin (10 μg/mL). Images of vessels were obtained with a multiphoton microscope 5 minutes before and after 17β-estradiol and then every hour for 4 hours. Representative three-dimensional images of each group at 4 hours of incubation; scale bar = 50 μm. Data are expressed as means ± standard error of the mean. n = 3 to 4 per group. *P ≤ 0.05 EC-ERαKO vs ERαFl₂.

Figure 6.

IEL characteristics of mesenteric arteries from ERαFl₂ and EC-ERαKO male mice fed a Western diet. (a and b) Representative confocal images of the IEL in mesenteric arteries; scale bar = 20 μm. (c and d) Number of fenestrae and area per fenestra (pixels) within the IEL in mesenteric arteries. (e) Modulus of elasticity specifically normalized as a function of the percolation of the IEL and its fenestrae. Data are expressed as means ± standard error of the mean. n = 4 to 5 per group. *P ≤ 0.05 EC-ERαKO vs ERαFl₂.

Discussion

In the current study, we used a male rodent model with absence of ERα in ECs. The main findings of our study are that after 20 weeks of WD feeding, loss of ERα signaling in ECs results in greater weight gain and outward hypertrophic remodeling in mesenteric arteries with increased deposition of extracellular matrix. This was paralleled with a decreased in MPP activation and an increase in the number of IEL fenestrae. In addition, and in an apparent paradoxical manner (as ERα signaling is classically considered protective in the vasculature), we demonstrated EC-ERαKO decreased stiffness in both aorta and mesenteric arteries of male mice fed a WD. These findings highlight an important role of ERα in modulating vascular remodeling in obese insulin-resistant males.

To the best of our knowledge, this is the first study to examine the role of endothelial ERα in males under conditions of overnutrition. The specificity of our model is driven by the selective expression of VE-cadherin in ECs during adult life (19). Previous seminal investigations ranging from preclinical models to humans have examined the role of estrogen, and more specifically ERα signaling, in the vasculature of males with differing results (14, 24, 25). Human evidence supports a role of ERα signaling in the maintenance of vascular homeostasis (24). A young man with a truncated form of ERα had premature coronary artery disease accompanied with an impaired endothelial-dependent dilatory response (32). In male porcine epicardial coronary arteries, ERα stimulation resulted in vasodilatory responses of similar magnitude to the ones seen in females (33). Rubanyi et al. (15) reported higher expression of ERα in aorta of C56Bl6/J male mice when compared with females. In the same investigation, the male ERα global KO mice exhibited decreased basal endothelial production of NO but no difference in ACh-induced dilation (15). The global ERαKO mice used in the later investigation are known to express isoforms of ERα, product of alternative splicing which has been postulated to explain residual estrogenic vascular effects (34, 35). Other investigators have evaluated the role of estrogen in the vasculature by eliminating the conversion of androgen to estrogens. Aromatase KO male mice have a blunted endothelial-dependent dilation with a normal endothelium-independent dilation response (16). Similarly, Lew et al. (17) demonstrated that 6 weeks of aromatase inhibition in healthy men resulted in an impairment of flow-mediated dilation without significant changes in arterial stiffness. In the present investigation, we showed that an absence of endothelial ERα does not result in differences in endothelial-dependent dilation in the setting of a high-fat and high-fructose feeding. Further, aortic endothelial-independent dilation was enhanced in the EC-ERαKO when compared with control. This latter finding may be related to increased VSMC sensitivity in the setting of chronic NO depletion (13); however, measures of NO were not performed in the current study. If that were the case, our finding that relaxation responses to the endothelium-dependent vasodilator ACh were no different between the cohorts may suggest that EC-ERαKO aortae produce less endothelial NO and have an enhanced VSMC response to compensate for this endothelial dysfunction. This potential explanation of our results remains to be tested experimentally.

In the current investigation, we also showed that absence of ERα in ECs of WD-fed males was associated with outward hypertrophic remodeling in mesenteric resistance arteries. Similar remodeling has been previously reported in a male rodent model of overnutrition (36) and has been related to increased mesenteric blood flow. Of note, in our investigation the EC-ERαKO did gain more weight than the controls also fed a WD, which might contribute to the outward hypertrophic remodeling. A previous study in females examined the role of ERα in a model of high flow-related remodeling in mesenteric vessels and reported an indispensable role of ERα−mediated eNOS activation for the occurrence of arterial remodeling. The apparent divergent results might be related to the fact that the latter findings were described in a nonobese female model in which the endothelial KO was driven by Tie2-Cre, which can also impact expression of the target gene in myeloid lineage (37).

Our current work also demonstrated that EC-ERαKO has increased extracellular deposition of collagen and elastin. Estrogens have been shown to modulate extracellular matrix remodeling via increased vascular expression of MMP (38, 39) and decreased expression of tissue inhibitors of MMP (40, 41). In a model of carotid injury, lack of estrogen was associated with decreased expression of MMP-2 and MMP-9. Similarly, in the current work we found that decreased endothelial ERα results in diminished gelatinolytic MMP activity upon stimulation with estradiol. Hence, our finding of increased collagen and elastin content in the vessel wall can also be related to the absence of ERα. Unexpectedly, in the present work, the remodeling paralleled an increase in arterial compliance as well as a decrease in arterial stiffness; these paradoxical changes may be attributed to a lack of change in the collagen to elastin ratio as the elastic modulus of collagen is greater than the one for elastin (42, 43). We found that the absence of endothelial ERα was associated with a higher number of fenestrae and a parallel decrease in their size. We have previously reported in a female model of exercise and overnutrition that the increased number and size of fenestrae in the femoral artery was associated with decreased arterial stiffness (29). Nevertheless, our current findings suggest that the fenestrae number may be more relevant to reducing stiffness than their size. In this regard, we hypothesize that the increase in the number of fenestrae in this model can facilitate a better diffusion of substances between the endothelium and the VSMCs. Notably, our results also emphasize a specific role of endothelial ERα in the control of arterial wall remodeling. Mutations limited to ECs have been previously associated with vascular remodeling (44), highlighting the important role of ECs in the maintenance of a physiological arterial wall remodeling.

As previously mentioned, in our current work we found that endothelial deletion of ERα in WD-induced obese males results in decreased stiffness in aorta. Arterial stiffness has been characterized as a deleterious phenomenon associated with aging and accelerated in clinical conditions such as obesity, type 2 diabetes, and insulin resistance (45–47). Augmented arterial stiffness results in increased risk of stroke, heart failure with preserved ejection fraction, chronic kidney disease, and cardiovascular death (46, 48). In the aorta, the finding of decreased arterial stiffness was not accompanied by changes in media thickness but there was a trend toward an increase in elastic fibers content. Thus, the finding of decreased arterial stiffness points toward a deleterious effect of endothelial ERα signaling in conditions of insulin resistance and overnutrition.

In the present investigation, we also reported that the endothelial-specific ERαKO mice gained more weight than the littermate controls under the same feeding conditions, with a trend toward greater insulin resistance. This result suggests that endothelial ERα is involved in regulation of energy homeostasis. ERα has been repeatedly shown to be involved in energy homeostasis, appetite control and physical activity regulation (49–51). Even though the main objective of this study was not focused on energy homeostasis, this interesting finding should be followed up in future metabolic studies. In the present investigation, we also reported that estradiol levels were almost twofold higher than previous levels reported in a global ERαKO (52). Therefore, it is possible that the elevated estradiol in our WD-fed model is related to weight gain (53). In light of these elevated levels of estradiol, it is conceivable that other estrogen receptors are being activated and can potentially exert vascular effects.

There are some important limitations in this study. ERα mRNA expression in ECs isolated from our EC-ERαKO mice was reduced by approximately 70%. This residual expression of ERα likely underestimates the role of EC-ERα in our model. Indeed, it is possible that in the setting of a complete deletion the detected effects would be magnified. Moreover, the incomplete deletion can also represent non-EC contamination of primary EC cultures. The residual expression of ERα is consistent with other KO models that have used the VE-cadherin promoter (54, 55). Another limitation of the current investigation is that our results are not directly comparable with our recently completed worked in females (14). We fed the males for a longer period of time than in our previous study, because we found that 8 weeks of WD was not sufficient to cause cardiovascular stiffness (20). An additional limitation of this study is that we did not assess blood pressure in the two cohorts. Our group and others have previously shown (50, 56) that under basal conditions a global KO of ERα does not result in blood pressure elevation despite weight gain. Nevertheless, ERα is known to have a central role in angiotensin II–induced hypertension (57) and future studies will be undertaken to explore the effect of EC- ERα in the modulation of blood pressure in the setting of insulin resistance and overnutrition.

In summary, we have demonstrated that deletion of endothelial ERα in males fed a diet high in fructose and fat has significant effects on the function and elasticity of their aortae and results in outward hypertrophic remodeling with reduced arterial stiffness in their mesenteric resistance arteries.