Background
Epidemiologic studies have demonstrated an association between endometriosis and the subsequent development of cardiovascular disease. The direct effect of endometriosis on the progression of atherosclerotic, if any, has not been previously characterized. Endometriosis leads to systemic inflammation that could have consequences for cardiovascular health. Here, we reported the effects of endometriosis on the development of atherosclerosis in a murine model.
Objective
This study aimed to determine the contribution of endometriosis in promoting cardiovascular disease in a murine model of endometriosis.
Study Design
Endometriosis was induced in 18 apolipoprotein E–null mice, the standard murine model used to study atherosclerosis. Mice of the same strain were used as controls (n=18) and underwent sham surgery without inducing endometriosis. The formation of endometriotic lesions was confirmed after 25 weeks of induction. Atherosclerotic lesions were subjected to hematoxylin and eosin staining followed by measurement of the aortic root luminal area and wall thickness. The whole aorta was isolated, and Oil Red O staining was performed to quantify the lipid deposits or plaque formation; moreover, biochemical assays were carried out in serum to determine the levels of lipids and inflammatory-related cytokines.
Results
Apolipoprotein E mice with endometriosis exhibited increased aortic atherosclerosis compared with controls as measured using Oil Red O staining (7.9% vs 3.1%, respectively; P =.0004). Mice with endometriosis showed a significant 50% decrease in the aortic luminal area compared with sham mice (0.85 mm 2 vs 1.46 mm 2 ; P =.03) and a significant increase in aortic root wall thickness (0.22 mm vs 0.15 mm; P =.04). There was no difference in the lipoprotein profile ( P <.05) between mice with endometriosis and sham mice. The serum levels of inflammatory cytokines interleukin 1 alpha, interleukin 6, interferon gamma, and vascular endothelial growth factor were significantly ( P <.05)increased in the endometriosis mice.
Conclusion
Our study used a murine model to determine the effect of endometriosis on atherosclerosis. Inflammation-related cytokines interleukin 1 alpha, interleukin 6, interferon gamma, and vascular endothelial growth factor (angiogenic factor) released by endometriotic lesions may contribute to the increased cardiovascular risks in women with endometriosis. To reduce the risk of cardiovascular disease, early identification and treatment of endometriosis are essential. Future treatments targeting inflammatory cytokines may help reduce the long-term risk of cardiovascular disease in women with endometriosis.
Introduction
Endometriosis is an estrogen-dependent inflammatory gynecologic disorder characterized by the growth of endometrial tissue outside the uterus that can cause chronic pelvic pain and infertility. Endometriosis affects 6% to 10% of reproductive-aged women, and the lesions are most typically found in the pelvic cavity. , Although endometriosis originates as a localized syndrome, the process of inflammation proves to be systemic, , where inflammatory cytokines, such as C-reactive protein (CRP), interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) , and vascular endothelial growth factor (VEGF), are found to be elevated in both the serum and the peritoneal fluid of women with endometriosis. ,
Why was this study conducted?
Endometriosis has long been considered a gynecologic disease; however, it has recently been demonstrated to be associated with other systemic illnesses, including cardiovascular disease. Women with endometriosis are typically lean and lack obvious risk factors for cardiovascular disease; it is unknown if the epidemiologic association with cardiovascular disease is directly related to endometriosis or because of other cofounders, including drug effects and surgical treatments. Determining a link between endometriosis and cardiovascular disease can identify and treat a previously unidentified at-risk patient population.
Key findings
Our study established endometriotic lesion formation in apolipoprotein E–null mice as evidenced by histology and identified more severe plaque development in the aortas of mice with endometriosis than controls using Oil Red O stain. Histology studies with hematoxylin and eosin staining revealed thickening of the aortic root wall and decreased luminal area in mice with endometriosis. Biochemical analysis revealed no change in lipid profile, although inflammatory-related cytokines and angiogenic factor levels were significantly increased in mice with endometriosis.
What does this add to what is known?
Our study demonstrated increased arterial plaque formation with endometriosis using a murine model. Endometriosis did not affect the lipid profile but did increase the production of inflammatory-related cytokines known to promote atherosclerosis. We identified atherosclerosis as a possible sequela of endometriosis, and we identified several novel targets to reduce the risk of cardiovascular disease in women with endometriosis.
More recently, endometriosis has been associated with an increased risk of multiple adverse health conditions, including cardiovascular disease, adverse reproductive outcomes, autoimmunity, endocrine disorders, and several cancers. , Epidemiologic studies have specifically identified increased cardiovascular risk in women with endometriosis; however, the mechanism underlying this has not yet been determined. The risk may be indirect and linked to loss of estrogen production resulting from oophorectomy or suppressive effects of medical therapies. Alternatively, cardiovascular disease may be the result of direct or indirect effects of endometriosis on vasculature. Atherosclerosis is a complex inflammatory process involving the interface of lipoproteins, monocyte-derived macrophages, and T cells with the vessel wall. , Women with endometriosis show significant elevations in markers of endothelial inflammation and activation. , Furthermore, recent evidence suggests higher oxidative stress and an atherogenic lipid profile in women with endometriosis, which may all contribute to the development of cardiovascular disease.
Hyperlipidemia has long been considered the major risk factor for the development of atherosclerosis. However, in several animal models of atherosclerosis, inflammation was shown to play an essential role in the pathogenesis of this disease, mediating all its stages and driving lipid accumulation in the intima of arteries. Multiple inflammatory markers, including CRP, serum amyloid A, IL-6, and TNF-α, are elevated in cardiovascular diseases, and the degree of elevation is associated with worse prognosis. Furthermore, endometriosis is associated with systemic inflammation and an increased number of activated macrophages. Hence, both disease processes share similar pathophysiological mechanisms.
Under normal conditions, mice do not develop atherosclerosis; therefore, apolipoprotein E (ApoE)-null mice are required in research settings. ApoE is a ligand that is crucial for the absorption and removal of atherogenic lipoproteins. In the absence of ApoE, mice develop hypercholesterolemia and early atherosclerotic lesions. Here, we reported the development of severe atherosclerosis in a murine model of endometriosis because of increased inflammatory-related cytokines.
Materials and Methods
Animals
ApoE-null c57BL/6 female mice aged 6 to 8 weeks were obtained from the Jackson Laboratory (Bar Harbor, ME) in compliance with an approved Yale Institutional Animal Care and Use Committee. All animals received a chow diet ad libitum. All in vivo experiments were carried out following Reporting of In Vivo Experiments guidelines. After 9 weeks of age, mice were randomly divided into 2 groups (n=18 per group), and endometriosis was induced and sham surgeries were carried out in the second group as control. These experiments were conducted in 2 separate replicates consisting of 10 experimental animals and 10 controls in the first set and 8 per group in the second set of experiments.
Induction of endometriosis in apolipoprotein E mice
Briefly, both uterine horns were extracted from donor ApoE mice by laparotomy. Uterine horns were separated, and each horn was opened longitudinally and sectioned horizontally, resulting in a total of 4 sections per uterus. Experimental recipient mice (n=18) were anesthetized using inhalation of isoflurane (2.5 L/min) in conjunction with oxygen (1.5 L/min). Moreover, 2 uterine segments were sutured on either side of the parietal peritoneum of each experimental mouse using 5-0 polyglactin suture (Vicryl), approximately 1 cm apart. The peritoneum and skin of the experimental mouse were closed with a 4-0 polyglactin suture. Sham surgeries were performed for the control group using the same surgical procedure without the introduction of donor uterine tissue (n=18). The experimental mice were allowed to develop endometriosis for 25 weeks. The development of the model was confirmed after transplantation via laparotomy and visualization of endometriotic lesions. After 25 weeks after transplantation and after an overnight fast, the mice were euthanized, and the endometriotic lesions were removed. Plasma was collected via cardiac puncture and stored at −80°C preceding analysis. Whole aortas were perfused and dissected from the 3 arches at the aortic arch to the 2 branches of the iliac arteries and then stored in formalin at 4°C before being subjected to Oil Red O (ORO) staining.
Hematoxylin and eosin staining
Lesions collected from mice with endometriosis were fixed in 5% paraformaldehyde overnight and transferred to 70% ethanol the next day and then paraffin embedded and sectioned into 50-μm thick sections using a vibratome. Hematoxylin and eosin (H&E) staining was followed by histologic examination for the confirmation of endometriosis in the ectopic lesions.
Oil Red O staining
ORO was purchased from VWR Life Science (Solon, Ohio; catalog number 0684-100G). ORO staining solution (weight-to-volume ratio, 0.25) was prepared with 35 mL ORO solution in methanol mixed with 10 mL of 1M sodium hydroxide and filtered with filter paper. Whole aortas were washed in 1 mL 78% methanol for 5 minutes on tilted roller paper, incubated in 1 mL of ORO staining solution for 50 minutes on tilted rollers, destained for 5 minutes in 1 mL 78% methanol, and stored in phosphate-buffered saline until ready for mounting and imaging.
Remnant adventitial fat was carefully removed using fine forceps under an Olympus SZX16 microscope (Olympus, Bloomfield, CT). Using microdissecting spring scissors, the aortas were cut longitudinally and pinned flat with the lumen side up onto clear-bottom Sylgard-coated glass dissecting dishes, as described. The images were captured using a Leica DFC295 microscope with a 10450528 0.5× XPF objective lens connected to an Olympus Model U-LH100HG camera at 0.73× with no adjustment to contrast or sharpness. A red threshold was used to analyze the images, determined based on the ability to visualize red stains in regions of plaque formation, using ImageJ software (Rasband, W.S., ImageJ, MD).
Histology of aortic root
The aortic root and whole aorta were separated and mounted in optimal cutting temperature (OCT) compound and frozen at −80°C until sections were obtained. Sections (5-μM thickness) were stained with H&E for histologic studies to determine the lumen area and wall thickness using ImageJ software.
Lipid profile analysis
Serum was collected via cardiac puncture after the mice were anesthetized. Blood was allowed to clot for 1 hour at room temperature, and the supernatant was collected after centrifugation at 5000 rpm for 10 minutes and stored at −80°C until used. Serum was analyzed for lipoprotein profile at Yale Core Center for Biochemical Assays (triglyceride [TG]; Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada), total cholesterol (TC), and high-density lipoproteins (HDL) (TC and HDL; Thermo Fisher Scientific, Waltham, MA). HDL was subtracted from TC to yield low-density lipoproteins (LDL).
Cytokine assay
Differential expression of cytokines in serum was determined using Mouse Cytokine/Chemokine 31-Plex Discovery Assay Array (catalog number MD31; Eve Technologies, Calgary, Canada). Experiments were carried out in duplicates and averaged for cytokines known to be associated with the development of atherosclerosis (IL1-α, IL-6, IFN-γ, and VEGF); furthermore, cytokines were plotted (endometriosis vs sham).
Statistical analysis
GraphPad Prism (GraphPad Software, La Jolla, CA) was used for statistical analyses of the data. Aortic plaque formation was quantified by ORO staining. Of note, 2 objective luminal areas and aortic wall thickness at equivalent anatomic locations were measured separately for each aorta on ImageJ software, and an average was taken of the resulting data. Moreover, t test or Mann-Whitney test was used to determine significant differences between the groups for quantitative plaque analyses, serum lipids, and serum cytokines.
Results
Lesion formation
Before aortic extraction, we examined the peritoneal cavity of the experimental (endometriosis) and sham (control) ApoE mice for endometriosis lesions at the implantation site or suture site. The mice in the group with endometriosis all had noticeable bilateral lesions at 25 weeks after transplantation of uterine tissue as shown in Figure 1 , A. The lesions were confirmed to be endometriosis using H&E staining that demonstrated growth of glandular and stromal endometrial tissue as shown in Figure 1 , B. No lesion was found in the corresponding location in control mice that underwent sham surgeries.
Plaque formation in aorta
To examine the effect of endometriosis on atherosclerotic plaque formation, we used ORO staining on the aortas with quantification of coloration threshold as displayed in Figure 2 , A. The ORO staining showed minimal plaque formation in the sham ApoE-null mice. In contrast, a large amount of plaque formation was noted in the ApoE-null mice with endometriosis. Plaque formation was significantly higher in mice with endometriosis than in sham (control) mice as shown in Figure 2 , B (mean±standard error of the mean [SEM]: 3.1%±0.9% in the control group and 7.9%±1.6% in the group with endometriosis; P =.0004), as determined by quantification of ORO coloration.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
