Alterations in Adhesion Molecules, Pro-Inflammatory Cytokines and Cell-Derived Microparticles Contribute to Intima-Media Thickness and Symptoms in Postmenopausal Women

<div><p>Menopause, the cessation of menses, occurs with estrogens decline, low-grade inflammation, and impaired endothelial function, contributing to atherosclerotic risk. Intima-media thickness (IMT) is an early subclinical biomarker of atherosclerosis. Inflammation may have a role on symptoms: hot flashes, anxiety, and depressive mood, which also are related to endothelial dysfunction, increased IMT and cardiovascular risk. In this study we compared several inflammatory markers in early <i>vs</i>. late postmenopausal women and studied the association of IMT and symptoms with these markers in the full sample. In a cross-sectional design including 60 women (53.1±4.4 years old) at early and late postmenopause, we evaluated the expression of CD62L, ICAM-1, PSGL-1, CD11b, CD11c, and IL-8R on PBMC by flow cytometry. Serum soluble ICAM-1, sVCAM-1, sCD62E, sCD62P, CXCL8, IL-1β, IL-6, and TNF-α levels were quantified by ELISA. Plasma levels of microparticles (MPs) were determined by FACS. Finally, carotid intima-media thickness (IMT) was measured by ultrasound. We observed that ICAM-1 expression by lymphocytes and serum sVCAM-1 levels were augmented at late postmenopause. Late postmenopause women with severe hot flashes had increased expression of CD62L and IL-8R on neutrophils. By multivariate analysis, the carotid IMT was strongly associated with membrane-bound TNF-α, CD11b expression, Annexin V⁺ CD3⁺ MPs, LPS-induced NO production, HDL-cholesterol and age. Depressive mood was associated negatively with PSGL-1 and positively with LPS-induced NO. Finally, Log(AMH) levels were associated with carotid IMT, IL-8R expression and time since menopause. IMT and depressive mood were the main clinical features related to vascular inflammation. Aging, hormonal changes and obesity were also related to endothelial dysfunction. These findings provide further evidence for a link between estrogen deficiency and low-grade inflammation in endothelial impairment in mature women.</p></div

Overexpression of ICAM-1 by lymphocytes and decreased of CD62L⁺ cells from late postmenopausal women.

<p>Mononuclear cells were isolated from the peripheral blood and were analyzed by flow cytometry for the adhesion molecules expression in lymphocytes gate. <b>a)</b> Single data points of ICAM-1 expression by lymphocytes are shown from both groups of postmenopausal women. ICAM-1 expression was significantly higher in late postmenopause women. Mean group values were compared by Student´s T test. <b>b-c)</b> Flow cytometry analysis of ICAM-1 expression on lymphocytes from early and late postmenopausal women. Histograms correspond to ICAM-1 staining from an early postmenopausal woman and a late postmenopausal woman. Percentages of positive cells and MFI values are indicated. <b>d-e)</b> Representative flow cytometry analysis percent of CD62L⁺ peripheral lymphocytes from an early and a late postmenopausal women are shown. Percentages of marked-positive cells are indicated. CD62L⁺ cells were significantly higher in early postmenopausal women. <b>f)</b> Data of percent of CD62L⁺ peripheral lymphocytes from early and late postmenopausal women are shown as mean±SD. (**<i>p</i><0.01; ***<i>p</i><0.001). MFI (mean fluorescence intensity).</p

CD62L and IL-8R expression is increased on neutrophils from postmenopausal women with hot flashes.

<p><b>a-b)</b> Neutrophils were isolated as stated in Material and Methods, then labeled for CD62L or <b>c-d)</b> IL-8R, and the percent of cells and MFI values were determined by flow cytometry analysis. Data are shown as the median and interquartile range. *<i>p</i><0.05, Kruskal-Wallis test and <i>post hoc</i> comparisons were carried out by Dunn´s test. Representative dot plots and histograms are shown. Red histograms correspond to CD62L <b>(b)</b> or IL-8R <b>(d)</b> staining. Percentages of positive cells and MFI values are indicated. MFI (mean fluorescence intensity).</p

Alterations in membrane-bound TNF-α-expression and percent of CD14⁺CD11c⁺ monocytes at late postmenopause.

<p>Mononuclear cells were isolated from the peripheral blood and immunostained for CD11c and membrane-bound TNF-<i>α</i> in CD14⁺monocytes gate, and analyzed by flow cytometry. <b>a-b)</b> Green histograms correspond to CD14⁺CD11c⁺ monocytes. Percentages of marker-positive cells are indicated. Data shown are representative of an early and a late postmenopausal woman. <b>c)</b> Percent of CD14⁺CD11c⁺ monocytes in early postmenopause was significantly higher than late postmenopause. Data shown as the mean±SD. *<i>p</i><0.05, Student´s T test. <b>d)</b> Comparison of membrane-bound TNF-<i>α</i> expression by monocytes from early and late postmenopausal women is shown as the mean±SD. Mean group values were compared by Student´s T test. (*<i>p</i><0.05).</p

Association of inflammatory markers with symptoms, carotid artery IMT, and hormones according to a multiple regression procedure.

<p>Multiple regression in all whole group (n = 60).</p><p><i>p</i><0.05 was considered statistical significant.</p><p>^*<i>p</i><0.05</p><p>^**<i>p</i><0.01</p><p>^***<i>p</i><0.001</p><p>Model #1 without and Model #2 with testing for confounding variables: group.</p><p>BMI, body mass index; AMH anti-Müllerian hormone; FSH, follicle-stimulating hormone; PSGL-1, P- and E-Selectin glycoprotein ligand-1; sTNF-α, soluble tumoral necrosis factor-alfa; NO, nitric oxide; LPS, lipopolysaccharide; IMT; intima media-thickness; Annexin V⁺CD3⁺ MPs, lymphocytes-derived MPs; Annexin V⁺ MPs, MP positive for phosphatidyl serine; S.E., standard error.</p><p>Association of inflammatory markers with symptoms, carotid artery IMT, and hormones according to a multiple regression procedure.</p

Possible effects of estrogens deprivation and aging on inflammation and endothelial damage at menopause.

<p>When estrogens decline and during old age, important changes occur in the endothelium: first, estrogen decrease, leads a low NO bioavailability inducing destabilization of endothelial cells, favoring up-regulation of adhesion molecules. Under such conditions, aged vessels have a compromised vasodilation, inducing endothelial dysfunction, increased vascular resistance and impaired tissue perfusion. Moreover, the atherosclerosis process is accompanied by a low-grade inflammation, which could be favored by endothelial dysfunction. The recruitment of leukocytes into areas of inflammation is mediated by interacting sets of cell adhesion molecules (showed step by step in order of activation). T-cells and monocytes are the first cells infiltrating the arterial intima, and represent a hallmark of endothelial damage favoring the early stages of atherogenesis. In atherosclerosis, focal expression of key adhesion molecules, mainly triggered by plasma atherogenic lipoproteins, may also mediate the recruitment of mononuclear cells to the plaque. Among these adhesion molecules, ICAM-1, a protein of the Ig superfamily, and one of the ligands for LFA-1, has been suggested to play an important role in atherogenesis. Thus, the increased ICAM-1 expression detected in this study could be associated with an enhanced recruitment of monocytes and T lymphocytes. Finally, in advanced stages, immune cells function also decline, contributing to inflammation and vascular damage and triggering CVD risk. (Modified from González-Amaro R. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120990#pone.0120990.ref051" target="_blank">51</a>]). ICAM-1, soluble intercellular adhesion molecule-1; sVCAM, soluble vascular cell adhesion molecule-1; L-Selectin, soluble lymphocyte selectin; TNF-α, tumoral necrosis factor-alfa; IL-8R, interleukin-8 receptor; NO, nitric oxide.</p

Comparison of inflammatory biomarkers between early and late postmenopausal women.

<p>All variables are shown as mean ± SD. Means between groups were analyzed by T student test.</p><p>^<i>a</i> Statistics calculated on logarithmic transformed values.</p><p><i>p</i><0.05 was considered significant.</p><p>^*<i>p</i><0.05</p><p>^**<i>p</i><0.01</p><p>^***<i>p</i><0.001</p><p>Annexin V⁺MPs, MP positive for phosphatidyl serine; Annexin V⁺CD3⁺MPs, lymphocytes-derived MPs; Annexin V⁺CD14⁺MPs, monocytes-derived MPs; Annexin V⁺CD62E⁺MPs, endothelial-derived MPs; Annexin V⁺CD41⁺MPs, platelet-derived MPs; sICAM-1, soluble intercellular adhesion molecule-1; sVCAM, soluble vascular cell adhesion molecule-1; sP-Selectin, soluble platelet-selectin, sE-Selectin; soluble endothelial selectin; sL-Selectin, soluble lymphocyte selectin; PSGL-1, P- and E-Selectin glycoprotein ligand-1; TNF-<i>α</i>, tumoral necrosis factor-alfa; IL-8R, interleukin-8 receptor; ROS, reactive oxygen species; NO, nitric oxide; LPS, lipopolysaccharide; IMT; intima-media thickness; MFI, mean fluorescence intensity.</p><p>Comparison of inflammatory biomarkers between early and late postmenopausal women.</p

Serum levels of sVCAM-1 are increased in late postmenopause.

<p>sVCAM-1 levels were determined by ELISA in serum samples from early and late postmenopausal women. Data are shown as the median and interquartile range. (*<i>p</i><0.05).</p

Comparison of anthropometric data, hormones and symptoms between early and late postmenopausal women.

<p>All variables are shown as mean ± SD or percentage, as appropriate. Group means were compared by Student’s t test.</p><p>^<i>a</i> Statistics calculated on logarithmic transformed values.</p><p>^<i>b</i> Proportions were analyzed using chi-squared (<i>X</i>²) test.</p><p>^*<i>p</i><0.05</p><p>^**<i>p</i><0.01</p><p>^***<i>p</i><0.001.</p><p>Reference ranges at menopause: 17β-E2, <35pg/ml; FSH, 40–200 mIU/ml; morning cortisol, 6.2–38.1 nmol/L; evening cortisol, 0.6–4.9 nmol/L.</p><p>BMI, body mass index; HDL, high-density lipoprotein; AMH anti-Müllerian hormone; FSH, follicle-stimulating hormone; IMT; intima-media thickness.</p><p>Comparison of anthropometric data, hormones and symptoms between early and late postmenopausal women.</p

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity.

PURPOSE:Obesity results from excess energy intake over expenditure and is characterized by chronic low-grade inflammation involving circulating monocytes (Mo) and group 2 innate lymphoid cells (ILC2s) imbalance. We analyzed circulating Mo subsets and ILC2s percentages and β2-adrenergic receptor (β2AR) expression in lean and obese subjects, and the possible effect of hypocaloric restriction on these innate immune cells. METHODS:In 139 individuals aged 45 to 57 years, classified in 74 lean individuals (>18.9kg/m2 BMI <24.9kg/m2) and 65 with obesity (n = 65), we collected fasting blood samples to detect Mo subsets, ILC2s number, and β2AR expression by flow cytometry. Lipids, insulin, leptin, and acylated-ghrelin concentrations were quantified. Resting energy expenditure (REE) was estimated by indirect calorimetry. These measurements were repeated in obese subjects after 7-weeks of hypocaloric restriction. RESULTS:Non-classical monocytes (NCM) and β2AR expression on intermediate Mo (IM) were increased in obese individuals (p<0.001, in both cases), whereas the percent of ILC2s was decreased (p<0.0001). Stepwise regression analysis showed significantly negative associations of ILC2s with caloric intake, β2AR expression on IM with REE, but a positive relationship between NCM and HOMA-IR. Caloric restriction allowed a significant diminution of NCM and the β2AR expression on IM, as well as, an increase in the percent of classical Mo (CM), and ILC2s. ΔREE was related to ΔCD16+/CD16- ratio. CONCLUSIONS:These findings show that in obesity occur changes in NCM, ILC2s and β2AR expression, which contribute to the low-grade inflammation linked to obesity and might revert with caloric restriction