Polycystic Ovary Syndrome Fuels Cardiovascular Inflammation and Aggravates Ischemic Cardiac Injury

Abstract

Background: Reducing cardiovascular disease burden among women remains challenging. Epidemiologic studies have indicated that polycystic ovary syndrome (PCOS), the most common endocrine disease in women of reproductive age, is associated with an increased prevalence and extent of coronary artery disease. However, the mechanism through which PCOS affects cardiac health in women remains unclear.

Methods: Prenatal anti-Müllerian hormone treatment or peripubertal letrozole infusion was used to establish mouse models of PCOS. RNA sequencing was performed to determine global transcriptomic changes in the hearts of PCOS mice. Flow cytometry and immunofluorescence staining were performed to detect myocardial macrophage accumulation in multiple PCOS models. Parabiosis models, cell-tracking experiments, and in vivo gene silencing approaches were used to explore the mechanisms underlying increased macrophage infiltration in PCOS mouse hearts. Permanent coronary ligation was performed to establish myocardial infarction (MI). Histologic analysis and small-animal imaging modalities (eg, magnetic resonance imaging and echocardiography) were performed to evaluate the effects of PCOS on injury after MI. Women with PCOS and control participants (n=200) were recruited to confirm findings observed in animal models.

Results: Transcriptomic profiling and immunostaining revealed that hearts from PCOS mice were characterized by increased macrophage accumulation. Parabiosis studies revealed that monocyte-derived macrophages were significantly increased in the hearts of PCOS mice because of enhanced circulating Ly6C⁺ monocyte supply. Compared with control mice, PCOS mice showed a significant increase in splenic Ly6C⁺ monocyte output, associated with elevated hematopoietic progenitors in the spleen and sympathetic tone. Plasma norepinephrine (a sympathetic neurotransmitter) levels and spleen size were consistently increased in women with PCOS when compared with those in control participants, and norepinephrine levels were significantly correlated with circulating CD14⁺⁺CD16^- monocyte counts. Compared with animals without PCOS, PCOS animals showed significantly exacerbated atherosclerotic plaque development and post-MI cardiac remodeling. Conditional Vcam1 silencing in PCOS mice significantly suppressed cardiac inflammation and improved cardiac injury after MI.

Conclusions: Our data documented previously unrecognized mechanisms through which PCOS could affect cardiovascular health in women. PCOS may promote myocardial macrophage accumulation and post-MI cardiac remodeling because of augmented splenic myelopoiesis.

Keywords: atherosclerotic plaque; immunity; macrophages; monocytes; myocardial infarction; polycystic ovary syndrome.

Figure 1.

Characterization of reproductive and metabolic phenotypes in a polycystic ovary syndrome mouse model. A, Schematic illustration showing the induction of polycystic ovary syndrome (PCOS) by prenatal anti-Müllerian hormone (PAMH) treatment. B, Serum testosterone levels in indicated groups (8 mice/group). C, Histologic analysis of ovaries. D, Number of corpora lutea in indicated groups (8 mice/group). E, PAMH mice exhibited acyclicity compared with control female mice. F, Body weight in indicated groups (8 mice/group). G, Body fat composition measured by dual-energy X-ray absorptiometry in indicated groups (8 mice/group). H, Representative images of hematoxylin & eosin staining of parametrial fat in indicated groups. I, Adipocyte cell sizes in indicated groups (100 cells from 5 mice/group). J, Parametrial fat weight in indicated groups (8 mice/group). K and L, Glucose tolerance test and insulin tolerance test results in indicated groups (8 mice/group). M, Serum lipid levels in indicated groups (8 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by Student t test (for D, F, G, J, K, L, and M), Mann-Whitney U test (for B), or a linear mixed model (for I). AUC indicates area under the curve; E, estrous stage; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; M/D, metestrus/diestrus stage; P, proestrous stage; TC, total cholesterol; and TG, triglycerides.

Figure 2.

Hearts of polycystic ovary syndrome mice are characterized by increased macrophage accumulation. A, Volcano plot of the RNA sequencing results obtained from hearts of polycystic ovary syndrome (PCOS) mice and control mice (5 or 6 mice/group). The red dots represent significantly upregulated genes (nominal P<0.05; fold change ≥2) and the blue dots represent significantly downregulated genes (nominal P<0.05; fold change ≤0.5). To avoid skewing of the plot, genes with >16-fold downregulation were omitted from the plot. B, Heatmap plot showing the expression levels of the top 10 upregulated genes in PCOS hearts. C, Quantitative polymerase chain reaction analysis results validated the upregulated genes in RNA sequencing analysis (5 mice/group). D through G, Results of the gene set enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) enrichment analysis of the RNA sequencing data. H, t-Distributed stochastic neighbor embedding plots showing that the top upregulated genes in PCOS mouse heart were enriched in the cardiac macrophage cluster. I, Flow cytometry analysis indicated that cardiac macrophages were significantly increased in hearts of PCOS mice (8 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by Student t test (for I); Student t test or Mann-Whitney U test was used for different genes in C.

Figure 3.

Circulating monocytes contribute to increased macrophage accumulation in hearts of polycystic ovary syndrome mice. A, 5-Bromodeoxyuridine (BrdU)⁺ incorporation in heart macrophages in indicated groups measured at 2 hours after BrdU injection (8 mice/group). B, Illustration showing the parabiosis models joining CD45.1 mice and CD45.2 mice to assess the contribution of circulating monocytes to increased cardiac macrophages in polycystic ovary syndrome (PCOS) mice. C, Flow cytometry analysis indicated that the chimerism of Ly6C⁺ monocytes in peripheral blood was comparable between PCOS pairs and control pairs (8 mice/group). D, The chimerism of donor-derived CD45.1⁺ cardiac macrophages was significantly increased in the PCOS parabiotic pairs compared with control pairs (8 mice/group). E and F, The parabiosis models joining ZsGreen⁺ mice and ZsGreen⁻ mice were used to confirm the contribution of circulating monocytes to increased cardiac macrophages in PCOS mice. Immunofluorescence staining results indicated that ZsGreen⁺ monocyte-differentiated cardiac macrophages were significantly increased in PCOS mice versus control mice (5 mice/group). G, Dot plots and quantification of Ly6C⁺ monocytes in the blood of PCOS and control mice (8 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by the Student t test (for A, D, F, and G) or Mann-Whitney U test (for C).

Figure 4.

Hematopoietic stem and progenitor cells are increased in the spleen of polycystic ovary syndrome mice. A and B, Dot plots and quantification of hematopoietic stem and progenitor cells (lineage⁻c-kit+Sca-1+ cells [LSK] and granulocyte macrophage progenitors [GMPs]) in the spleen of polycystic ovary syndrome (PCOS) and control mice (8 mice/group). C, Spleen weight in indicated groups (8 mice/group). D, Quantification of GMPs in the blood of PCOS and control mice (8 mice/group). E and F, Dot plots and quantification of CD45.1⁺ partner-derived splenic GMP cells in PCOS pairs and control pairs (8 mice/group). G, Representative images and quantification of tyrosine hydroxylase staining in the bone marrow of PCOS mice and control mice (5 mice/group). H, Dot plots showing GMP (in the bone marrow, blood, and spleen), Ly6C⁺ monocytes (in the spleen and blood), and cardiac macrophages in indicated groups. I through K, Quantification of GMPs in the bone marrow, blood, and spleen in indicated groups (8 mice/group). L through N, Quantification of Ly6C⁺ monocytes in the spleen and blood and cardiac macrophages in indicated groups (8 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by Student t test (for B, C, F, and G) or Mann-Whitney U test (for D) or 2-way ANOVA with Tukey multiple comparisons test (for I, J, K, L, M, and N). Log transformation was done for blood GMP numbers to pass the heteroscedasticity test. 6-OHDA indicates 6-hydroxydopamine hydrobromide.

Figure 5.

Cardiac inflammation and ventricular remodeling after myocardial infarction are augmented in polycystic ovary syndrome mice. A through D, Dot plots and quantification of monocyte, macrophage, and neutrophils in the myocardium of polycystic ovary syndrome (PCOS) mice and control mice at 3 days after myocardial infarction (MI; 10 mice/group). E, Immunofluorescence staining and quantification of macrophages and neutrophils in infarcted hearts from PCOS mice and control mice (5 mice/group). F through H, Transcriptional levels of proinflammatory cytokines in indicated groups (10 mice/group). I and J, Myocardial apoptosis in the infarct border zone was measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining (5 mice/group). K, Representative images of Masson trichrome staining in indicated groups at 3 weeks after MI. L, Quantification of scar circumference and scar thickness in indicated groups at 3 weeks after MI (10 or 12 mice/group). M, Representative images of small animal echocardiography and quantitative analysis of left ventricular ejection fraction (LVEF) in indicated groups at 3 weeks after MI (10 or 12 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by Student t test (for B, D, E, G, H, I, L, and M); Mann-Whitney U test was used for C and F. IL1β indicates interleukin-1β; IL6, interleukin-6; and TNFα, tumor necrosis factor–α.

Figure 6.

Atherosclerotic plaque development is accelerated in polycystic ovary syndrome mice. A, Representative images of the plaque sections stained with hematoxylin & eosin (H&E), Masson trichrome, or oil red O. B, Quantification of the atherosclerotic lesion size, necrotic core size, lipid content, and fibrous cap thickness in indicated groups (20 mice/group). C through F, Dot plots and quantification of intraplaque monocytes, macrophages, and neutrophils measured by flow cytometry in indicated groups (10 mice/group). G, Immunofluorescence staining of intraplaque macrophages and neutrophils in indicated groups (5 mice/group). H through K, Transcriptional levels of inflammatory markers in indicated groups (8 mice/group). Data are expressed as mean±SEM. Statistical analysis was performed by the Student t test (for B, D, F, and J) or Mann-Whitney U test (for E, H, and I). Ly6C indicates lymphocyte antigen 6 complex, locus C1; and PCOS, polycystic ovary syndrome.

Figure 7.

Illustrations of the mechanisms underlying the negative cardiovascular effects of polycystic ovary syndrome. Polycystic ovary syndrome (PCOS) mobilized hematopoietic progenitor cells in the bone marrow, leading to increased circulating hematopoietic progenitor cells; meanwhile, splenic norepinephrine content is increased in PCOS mice, contributing to increased expression of a hematopoietic progenitor retention factor Vcam1 (vascular cell adhesion molecule 1) in splenic macrophages, subsequently leading to increased splenic retention of hematopoietic progenitor cells and monocytopoiesis; increased circulating Ly6C⁺ monocyte supply enhanced inflammatory burden in heart and atherosclerotic plaques, promoting remodeling after myocardial infarction (MI) and atherosclerotic plaque instability.