Abnormally high follicle-stimulating hormone (FSH) has been reported to associate with cardiovascular diseases in prostate cancer patients with specific androgen deprivation therapy and in menopausal women. All of the cardiovascular diseases were involved in atherosclerosis. However, the pathogenic mechanism of FSH-associated atherosclerosis remains uncertain. Apolipoprotein E–deficient mice were chosen to develop atherosclerosis, of which the plaques were analyzed with administration of short- and long-term FSH imitating androgen deprivation therapy–induced and menopausal FSH elevation. The study showed that short- and long-term exposure of FSH significantly accelerated atherosclerosis progression in apolipoprotein E–deficient mice, manifested as strikingly increased plaques in the aorta and its roots, increased macrophage content, reduced fibrin, and an enlarged necrotic core, suggesting a decrease in plaque stability. Furthermore, expression profiles from the Gene Expression Omnibus GSE21545 dataset revealed that macrophage inflammation was tightly associated with FSH-induced atherosclerotic progression. The human monocyte cell line THP-1 was induced by PMA and worked as a macrophage model to detect inflammatory factors and cellular functions. FSH remarkably promoted the expression of IL-1β in macrophages and strikingly increased the chemotactic migratory capacity of macrophages toward MCP-1, but the promigratory capacity of FSH was attenuated in foam cells. Overall, we revealed that FSH significantly promoted the inflammatory response and migration of macrophages, thereby provoking atherosclerosis development.

Prostate cancer ranks second in the leading new cancer cases in men behind lung cancer (1). Androgen deprivation therapy (ADT) is mainly used to treat patients with advanced and metastatic prostate cancer (2). Although these treatments can bring prostate cancer under control, the adverse reactions, specifically fatal cardiovascular diseases (CVDs) (3), have resulted in ADT gradually attracting people’s attention.

ADT is divided into two main categories, that is, drugs (gonadotropin-releasing hormone [GnRH] agonists or antagonists) and surgical castration (4). Researchers have noticed that certain ADTs, such as GnRH agonists and surgical castration, are usually accompanied by significant fluctuations of follicle-stimulating hormone (FSH) (5). For surgical castration, a low androgen level can lead to a feedback increase of GnRH in serum, further stimulating the pituitary to secrete FSH and maintaining it at a higher level for a long time. GnRH agonists can temporarily activate the expression of GnRH receptors in pituitary cells and increase FSH level for ∼2 wk. When the GnRH receptors of pituitary cells are gradually desensitized, FSH level begins to decline. The fluctuations of FSH do not happen with GnRH antagonists. The main reason is that GnRH agonists mainly inhibit luteinizing hormone, whereas antagonists can inhibit FSH and luteinizing hormone (5). The high serum level of FSH seems a prominent difference between GnRH agonist and GnRH antagonist treatment. Based on these studies, we proposed the hypothesis that FSH is likely to play a key role in the adverse cardiovascular events caused by specific ADT treatments. Similarly, serum FSH levels also undergo dramatic elevation in menopausal females (6). Several studies have shown that FSH is closely related to cardiovascular disease in menopausal women (7).

For almost all of the adverse cardiovascular events, such as stroke (8), thrombosis (9), and myocardial infarction (10), atherosclerosis (AS) is a basic vascular disease and prerequisite. The lesions of AS are characterized by the gradual accumulation of plaques in large and medium-sized artery vessels (11). Macrophages are involved in various stages of AS development and are the main mediators of arterial plaque formation (12). Macrophages in various human tissues are mainly directly differentiated from monocytes in the blood and play an important role in the inflammatory and anti-inflammatory response in multiple chronic inflammatory diseases, such as AS (13).

FSH is a glycoprotein hormone, mainly secreted by pituitary cells, and exerts a physiological effect via binding to a specific FSH receptor (14). Recently, more and more studies have found that FSH receptors are not only present in reproductive organs such as testes and ovaries, but they also exist in immune cells (monocytes/macrophages and others) (15) and vascular endothelium (16). These studies suggest that FSH is involved in other pathological and physiological processes and that the relationship between AS and FSH has not been fully discovered, especially the effect of FSH on monocytes/macrophages. Therefore, we asked whether FSH promotes the development of AS by affecting the biological function of monocytes/macrophages, thereby mediating the occurrence of ADT-related cardiovascular complications. If so, what is the molecular mechanism? Based on the above problems, we carried out this study.

In this study, we used an AS animal model with apolipoprotein E–deficient (ApoE−/−) mice to initially prove that the short- and long-term exogenous FSH supplementation could accelerate AS progression. Furthermore, the plaque analysis and bioinformatics prediction showed that macrophages were significantly affected by FSH. In our in vitro study, FSH could active macrophages by stimulating the expression and secretion of IL-1β, promoting atherosclerotic plaque progression. Our investigation may provide some inspiration for future endocrine therapy of prostate cancer: to prevent adverse cardiovascular events during the ADT treatment, it is essential to control the FSH level and take into consideration cardiovascular risk, particularly in patients with existing cardiovascular diseases.

Eight-week-old homozygous ApoE−/− male mice on the C57BL/6 background were generated by the Peking University Health Science Center Department of Laboratory Animal Science and were fed a high-fat and high-cholesterol diet for 12 wk. Serum FSH levels were ∼1- to 20-fold higher in mice with castration/ovariectomy and GnRH agonist therapy (the early stage of treatment) (17, 18), on which we based FSH administration. FSH (R&D Systems, Minneapolis, MN) was s.c. administered to the ApoE−/− mice for 2 and 12 wk, respectively, with saline serving as the vehicle control. The 2-wk FSH treatment (FSH-2w) imitated the short-term escalation of FSH in GnRH agonist treated prostate cancer patients, the 12-wk FSH treatment (FSH-12w) imitated long-term increasing FSH both in menopausal women and prostate cancer patients with orchiectomy. At the two terminals of FSH administration, blood samples were taken and the serum was stored at −80°C. All animal procedures were approved by the Peking University People’s Hospital Animal Use Protocol and Ethics Review (reference no. 2020PHE096) and were performed according to the guidelines approved by Animal Care and Use Committee of Peking University.

In brief, after 12 wk of treatment, the mice were sacrificed and the heart and aortic tree were isolated carefully and fixed in 4% paraformaldehyde. The heart was embedded in NEG-50 (Thermo Fisher Scientific, Waltham, MA) and the aortic sinus was serially sectioned into 5-μm-thick cryosections and then stained with oil red O, Masson trichrome, or anti-F4/80 Ab (Cell Signaling Technology, Danvers, MA), anti-CD80 Ab (Abcam), and anti-CD163 Ab (Abcam) and DAPI (Thermo Fisher Scientific). The aortic root and aortic tree were stained by Oil Red O.

Total plasma of the ApoE−/− mice fed and treated as indicated was assayed for cholesterol and triglyceride concentrations using commercially available kits based on the Trinder chromogenic reaction (19) (Biosino, Beijing, China). The ELISA kits (described below) were used for detection of plasma cytokines FSH and IL-1β.

Gene expression profiles of the GSE21545 dataset (made public on March 28, 2012; last updated May 5, 2022) were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/), in which 223 patients with AS were enrolled. The carotid atherosclerotic plaques and PBMCs were collected and analyzed. RNA was extracted and measured through microarray analysis (platform: GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array).

The Human Protein Atlas (THPA) database (http://www.proteinatlas.org/) (20) was used to evaluate the cell markers of PBMCs through single-cell transcriptomic data (21).

THP-1 cells (22) purchased from the American Type Culture Collection were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and incubated at 37°C with 5% CO2. The exponentially growing THP-1 cells were induced by PMA for 24 h, stably differentiated into macrophages after 48 h, and then treated with 50 ng/ml FSH (Aladdin Industrial, Shanghai, China) for 24 h.

Total RNA was extracted from PMA-induced THP-1 cells by the TRIzol method. Quantitative real-time PCR was applied to quantify transcriptional expressions and the internal control gene GAPDH using Hieff quantitative PCR SYBR Green master mix (Yeasen Biotechnology, Shanghai, China) and the following specific primers: IL-1β, 5′-ATGGCTTATTACAGTGGCAA-3′ (forward), 5′-TCAGCTTGTCCATGGCCACAA-3′ (reverse); Nos2, 5′-CTGGGCTACACTGAGCACC-3′ (forward), 5′-AAGTGGTCGTTGAGGGCAATG-3′ (reverse); IL-6, 5′-GCCCACCAAGAACGATAG-3′ (forward), 5′-GGTTGTCACCAGCATCAG-3′ (reverse); TNF-α, 5′-CACCATGAGCACAGAAAGCA-3′ (forward), 5′-TAGACAGAAGAGCGTGGTGG-3′ (reverse); MCP-1, 5′-GTGCTGACCCCAAGAAGGAATG-3′ (forward), 5′-TGAGGTGGTTGTGGAAAAGGTAGTG-3′ (reverse); claudin 12 (CLDN12), 5′-AGCAGTGACTGCCTGATGTACGA-3′ (forward), 5′-ATGGCGATCAGCATGCTGAGG-3′ (reverse); TJP1 (tight junction protein 1), 5′-AATGGATAATGTTGAACATGC-3′ (forward), 5′-ATCAGGACGACTTACTGGTAT-3′ (reverse); IL-10, 5′-CCGTGGAGCAGGTGAAGAAT-3′ (forward), 5′-TAGAGTCGCCACCCTGATGT-3′ (reverse); CD206, 5′-AACGGACTGGGTTGCT-3′ (forward), 5′-ATCCCTTGTAGAGCAT-3′ (reverse); CD209, 5′-AATGGCTGGAACGACGA-3′ (forward), 5′-CAGGAGGCTGCGGACTTTTT-3′ (reverse); and GAPDH, 5′-CTGGGCTACACTGAGCACC-3′ (forward), 5′-AAGTGGTCGTTGAGGGCAATG-3′ (reverse). Conditions of the PCR reaction were as follows: one cycle of 95°C for 5 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. Each reaction was repeated three times. The expression level of mRNA was determined by relative quantitative Ct value of gene expression with the 2–ΔΔCt method.

The total proteins were purified from the treated cells with RIPA buffer (Beijing Solarbio Science & Technology, Beijing, China) containing a protease inhibitor mixture (Thermo Fisher Scientific). The concentrations of total protein lysates were determined by an enhanced bicinchoninic acid protein quantification assay kit (Thermo Fisher Scientific). Next, proteins (20–30 μg per well) were used for 10% SDS-PAGE. Then, the proteins were transferred to polyvinylidene difluoride membranes, which then were blocked with 5% nonfat milk. All kinds of Abs were diluted by Ab dilution solution (New Cell & Molecular Biotech, Suzhou, China) and were incubated using the manufacturer’s instructions. Abs used were as follows: rabbit anti–IL-1β mAb (12703, Cell Signaling Technology), rabbit anti–inducible NO synthase (iNOS) mAb (20609, Cell Signaling Technology), rabbit anti-CLDN12 polyclonal Ab (38-8200, Thermo Fisher Scientific), mouse anti–β actin mAb (TA-09, ZSGB-BIO).

IL-1β, TNF-α, and IL-6 in a culture supernatant of PMA-induced THP-1 cells were detected by a human IL-1β ELISA kit, human TNF-α ELISA kit, and human IL-6 ELISA kit, respectively (Meimian Industrial, Jiangsu, China). The serum FSH and IL-1β in ApoE−/− mice were confirmed by a mouse FSH ELISA kit and mouse IL-1β ELISA kit (Cloud-Clone, Wuhan, China), respectively. The experiments were carried out according to the manufacturers’ instructions.

PMA-induced THP-1 cells were planted in the upper insert of a Boyden chamber at a concentration of 5 × 104 cells per well. The lower chamber was filled with 600 μl of RPMI 1640 medium containing 20% FBS and 5 ng/ml MCP-1. Following incubation at 37°C for 48 h, the cells on the lower surface of the filter membrane were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.5% gentian violet solution at room temperature for 20 min. The cells were imaged with a 10 × 20 optical microscope and counted in five random fields.

The proliferation of PMA/THP-1 under the indicated treatment was detected by a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Cells were planted into 96-well plates with a density of 3 × 104 cells per well. At the indicated time points, 10 μl of CCK-8 was added to each well and incubated at 37°C for 2 h. Subsequently, the absorbance of live cells was measured at 450 nm by a microporous plate reader.

PMA and THP-1 were starved overnight in RPMI 1640 medium (Life Technologies, Carlsbad, CA) containing 5.0% FBS and then incubated with 10 μg/ml oxidized low-density lipoprotein (ox-LDL; Bioss, Beijing, China) for 24 h. After washing away unspecific bound LDL, cells were stained with oil red O.

Data were analyzed using the GraphPad Prism 8.0 statistical software package. All cell biology experiments were repeated at least three times, and data were analyzed by an unpaired t test (two-tailed) and are presented as the mean ± SEM. One-way ANOVA was used to analyze the one-way difference between two or more groups. A significant statistical difference between groups is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Recently, clinical studies have reported that fluctuations of FSH in prostate cancer patients induced by ADT may be associated with AS (2325). Therefore, we first explored the effect of FSH on AS and applied FSH as an independent stimulus to ApoE−/− mice by which we constructed an AS disease model. The procedure of the animal experiment is shown in Supplemental Fig. 1A. We first verified the effect of s.c. administration of FSH on the concentration of serum FSH in mice and detected FSH level in blood by ELISA. The results showed that the concentration of FSH in the two groups treated with FSH exposure (FSH-2w and FSH-12w) was significantly higher than that in the control group (Fig. 1B), indicating that s.c. administration can continuously and steadily increase the serum FSH concentration. The results of oil red O staining showed that the area of plaque in the aortic tree was enlarged in mice with short- and long-term FSH exposure when compared with the saline group (Fig. 1A), showing that FSH could significantly increase the plaque area (Fig. 1B). Additionally, there was no significant difference in plaque area between the short-term and long-term FSH treatment groups (p = 0.1045). Furthermore, we analyzed the aortic plaques by oil red O, Masson trichrome, and immunofluorescence to observe plaques at the aortic root and the content of collagen fibers and macrophages in the plaques, comprehensively evaluating the stability of the plaques. The results showed that FSH exposure significantly increased the plaques at the aortic root (Fig. 1C, 1D) in which collagen fibers were significantly reduced (Fig. 1E) and macrophages clearly increased (Fig. 1F), and the plaque necrosis core was apparently enlarged, which suggested that FSH could promote AS progression and undermine the plaque stability.

FIGURE 1.

Analysis of plaques in ApoE−/− mice with the short- and long-term FSH exposure. ApoE−/− mice were injected s.c. with 6 mIU FSH per mouse or saline (n = 3 mice) every day for 2 wk (n = 4 mice) and 12 wk (n = 4 mice) and fed a high-fat and high-cholesterol diet throughout. Medial canthal venous blood samples were collected at 2 and 12 wk of administration. (A) Representative oil red O staining of aortic tree from ApoE−/− mice treated with saline, FSH (2 wk, FSH-2w), and long FSH (12 wk, FSH-12w). Scale bar, 1 cm. (B) ELISA for serum FSH concentration detection at 2 and 12 wk of FSH treatment. (C and D) Representative oil red O staining of aortic sinus of ApoE−/− mice treated with saline or FSH (FSH-2w and FSH-12w). Black arrows point to necrotic core. Scale bar, 250 μm. (E) Representative Masson staining of aortic sinus cryosections from ApoE−/− mice treated with saline or FSH (FSH-2w and FSH-12w). Black arrows point ot necrotic core. Scale bars, 100 μm. (F) Immunofluorescence staining with the F4/80 Abs in aortic sinus cryosections from ApoE−/− mice. DAPI was used to stain the nucleus. Scale bar, 50 μm. *p < 0.05, **p < 0.01. n.s., not significant.

FIGURE 1.

Analysis of plaques in ApoE−/− mice with the short- and long-term FSH exposure. ApoE−/− mice were injected s.c. with 6 mIU FSH per mouse or saline (n = 3 mice) every day for 2 wk (n = 4 mice) and 12 wk (n = 4 mice) and fed a high-fat and high-cholesterol diet throughout. Medial canthal venous blood samples were collected at 2 and 12 wk of administration. (A) Representative oil red O staining of aortic tree from ApoE−/− mice treated with saline, FSH (2 wk, FSH-2w), and long FSH (12 wk, FSH-12w). Scale bar, 1 cm. (B) ELISA for serum FSH concentration detection at 2 and 12 wk of FSH treatment. (C and D) Representative oil red O staining of aortic sinus of ApoE−/− mice treated with saline or FSH (FSH-2w and FSH-12w). Black arrows point to necrotic core. Scale bar, 250 μm. (E) Representative Masson staining of aortic sinus cryosections from ApoE−/− mice treated with saline or FSH (FSH-2w and FSH-12w). Black arrows point ot necrotic core. Scale bars, 100 μm. (F) Immunofluorescence staining with the F4/80 Abs in aortic sinus cryosections from ApoE−/− mice. DAPI was used to stain the nucleus. Scale bar, 50 μm. *p < 0.05, **p < 0.01. n.s., not significant.

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Additionally, compared with the control group, serum total cholesterol concentration was distinctly raised in the FSH long-term treatment group and increased in the FSH short-term treatment group (p = 0.0552) (Supplemental Fig. 1B). Also, there was no significant difference in serum triglyceride concentrations among the groups (saline versus FSH-2w, p = 0.3554; saline versus FSH-12w, p = 0.1995; FSH-2w versus FSH-12w, p = 0.9959) (Supplemental Fig. 1C). Comparing the monthly body weight changes of each group, there was no significant difference in the body weight at the four time points (8, 12, 16, and 20 wk old) respectively (Supplemental Fig. 1D). Furthermore, we have initially confirmed that FSH may improve atherosclerotic plaque development.

Macrophages are important members in AS plaque formation and lesion stability. According to the results of the above animal assay, we attempted to predict the relevance of FSH and macrophages and the potential factors using gene expression profiles of the GSE21545 dataset downloaded from Gene Expression Omnibus database. We compared the carotid plaque samples of 126 patients with their PBMCs (97 blood samples) and observed 22,174 differentially expressed genes containing 10,917 upregulated and 11,258 downregulated genes (Fig. 2A). Among these genes, NOS2, IL-1B, and IL-1R were related to macrophage activation and inflammation, and CLDN12 and TJP1were associated with vascular intima integrity and cellular motion (Fig. 2B). Correlation analyses revealed that five genes were highly correlated with FSH/FSHR (FSHB expresses the functional β subunit of FSH) (Fig. 2C). Functional enrichment analysis displayed that these five differentially expressed genes were enriched in macrophage activation, migration, and chemotaxis (Fig. 2D). THPA database contained single-cell transcriptomic data of PBMCs, and we identified the significant cell markers of macrophages as CD163, CD68, MARCO, MRC1, MSR1, and IL-1β (Fig. 2E). Based on these results, we explored the regulation of FSH on macrophage biological functions, such as the inflammatory phenotype and cellular migration.

FIGURE 2.

Analysis of GSE21545 for prediction of FSH-related factors. (A) Volcano plots of differentially expressed genes between the carotid plaque samples (n = 126 patients) and their PBMCs (n = 97 blood samples). (B) Heatmap of genes related to macrophage inflammation. (C) Correlation of FSHB/FSHR and CLDN12, IL-1B and IL-1R, NOS2, and TJP1. (D) Gene Ontology (GO) enrichment analysis indicates CLDN12, IL-1B and IL-1R, NOS2, and TJP1 were enriched in regulation of macrophage activation and motion process (GO:0042116), macrophage migration (GO:1905517), and leukocyte migration (GO:0050900). (E) IL-1β expression shown in single-cell transcriptomic data of PBMCs in THPA database. CLDN12, claudin 12; NOS2, NO synthase 2; TJP1, tight junction protein 1.

FIGURE 2.

Analysis of GSE21545 for prediction of FSH-related factors. (A) Volcano plots of differentially expressed genes between the carotid plaque samples (n = 126 patients) and their PBMCs (n = 97 blood samples). (B) Heatmap of genes related to macrophage inflammation. (C) Correlation of FSHB/FSHR and CLDN12, IL-1B and IL-1R, NOS2, and TJP1. (D) Gene Ontology (GO) enrichment analysis indicates CLDN12, IL-1B and IL-1R, NOS2, and TJP1 were enriched in regulation of macrophage activation and motion process (GO:0042116), macrophage migration (GO:1905517), and leukocyte migration (GO:0050900). (E) IL-1β expression shown in single-cell transcriptomic data of PBMCs in THPA database. CLDN12, claudin 12; NOS2, NO synthase 2; TJP1, tight junction protein 1.

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The macrophage pool in AS plaques is composed of a complex mix of macrophage subsets that exhibit dynamic plasticity in phenotype and function in response to various stimuli playing beneficial or detrimental roles in AS (26). In extreme cases, macrophages are phenotypically divided into inflammatory M1 macrophages and anti-inflammatory M2 macrophages. PMA can induce the differentiation of THP-1 into macrophages, which is a classic model for studying M0 macrophages, and furthermore IFN-γ and LPS can induce M0 macrophages to differentiate into M1 macrophages, and IL-4 can induce M0 macrophages to differentiate into M2 macrophages (27). NOS2, TNF-α, IL-1β, and IL-6 are the signature inflammatory factors secreted by M1 macrophages. CD206, CD209, and IL-10 are the signature anti-inflammatory factors secreted by M2 macrophages (28). We verified the biological effects of FSH on the phenotype of macrophages by detecting the signature factors at the mRNA level. The results showed that the inflammatory molecules NOS2, IL-1β, and IL-6 were significantly elevated at the mRNA level in M0 macrophages (PMA/THP-1), but not clearly changed in M1 macrophages, with FSH treatment (Fig. 3A–C). Meanwhile, MCP-1, TNF-α, and the anti-inflammatory molecules CD209, CD206, and IL-10 were apparently not changed in both M0 and M1/M2 macrophages (Fig. 3D–H) with or without FSH treatment. Additionally, the predicted molecule CLDN12 was remarkably increased in M0 macrophages with FSH exposure, but not TJP1 (Supplemental Fig. 2A, 2B). These results suggested that FSH may induce the inflammatory differentiation of macrophages, but barely promote the inflammatory response of M1 macrophages. Furthermore, the macrophage populations in plaques were analyzed by the M1 macrophage marker CD80 and the M2 macrophage marker CD163 (29). Data showed that FSH exposure strikingly promoted M1 macrophage infiltration and accumulation in plaques (Fig. 3I), contributing to plaque formation and instability. The M2 macrophages had no significant difference among the short-term and long-term FSH treatment and saline groups. These results indicated that FSH could provoke a proinflammatory differentiation of macrophages and the accumulation of M1 macrophages in plaques.

FIGURE 3.

Effects of FSH on signature factor expression in macrophages. THP-1 cells were induced by PMA for 24 h and then were treated with FSH (50 ng/ml) for 24 h; PMA-induced THP-1 cells were further treated with IFN-γ and LPS for 24 h or IL-4 for 24 h and then were treated with FSH for 24 h. (AE) Effects of FSH on expression of inflammatory molecules IL-1β, NOS2, IL-6, MCP-1, and TNF-α in M0 and M1 macrophages were determined by quantitative real-time PCR. (FH) Effects of FSH on expression of anti-inflammatory molecules CD209, CD206, and IL-10 in M0 and M2 macrophages were determined by quantitative real-time PCR. (I) Immunofluorescence staining with the F4/80 Abs and CD80 Abs in aortic sinus cryosections from ApoE−/− mice. Scale bar, 50 μm. (J) Immunofluorescence staining with the F4/80 Abs and CD163 Abs in aortic sinus cryosections from ApoE−/− mice. Scale bar, 50 μm. Data shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

FIGURE 3.

Effects of FSH on signature factor expression in macrophages. THP-1 cells were induced by PMA for 24 h and then were treated with FSH (50 ng/ml) for 24 h; PMA-induced THP-1 cells were further treated with IFN-γ and LPS for 24 h or IL-4 for 24 h and then were treated with FSH for 24 h. (AE) Effects of FSH on expression of inflammatory molecules IL-1β, NOS2, IL-6, MCP-1, and TNF-α in M0 and M1 macrophages were determined by quantitative real-time PCR. (FH) Effects of FSH on expression of anti-inflammatory molecules CD209, CD206, and IL-10 in M0 and M2 macrophages were determined by quantitative real-time PCR. (I) Immunofluorescence staining with the F4/80 Abs and CD80 Abs in aortic sinus cryosections from ApoE−/− mice. Scale bar, 50 μm. (J) Immunofluorescence staining with the F4/80 Abs and CD163 Abs in aortic sinus cryosections from ApoE−/− mice. Scale bar, 50 μm. Data shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

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Based on the results of transcriptional expression, we further examined the effect of FSH on the expressions of IL-1β, TNF-α, IL-6, and NOS2 at protein levels. IL-1β, TNF-α, and IL-6 are all secreted proteins, which were measured by ELISA. The results showed that IL-1β was significantly increased in the culture supernatant of FSH-treated PMA/THP-1 cells compared with that in the control group (Fig. 4A). However, the levels of TNF-α (p = 0.2877) and IL-6 (p = 0.9603) were not distinctly changed between the FSH and control groups (Fig. 4B, 4C). Meanwhile, the total expression of IL-1β was profoundly elevated with FSH exposure (Fig. 4F), consistent with the ELISA detection.

FIGURE 4.

Effects of FSH on protein expression levels of inflammatory cytokines in macrophages. PMA/THP-1 cells were treated with FSH (50 ng/ml) for 24 h and the culture supernatant or cells were collected. (AC) The concentration levels of IL-1β (A), TNF-α (B), and IL-6 (C) in the culture supernatant of PMA/THP-1 cells were assessed by ELISA. (D) The expression levels of iNOS in PMA/THP-1 cells were assessed by Western blot. (E) The concentration levels of NO in the culture supernatant of PMA/THP-1 cells were assessed. (F) The expression levels of IL-1β in PMA/THP-1 cells were assessed by Western blot. (G) Serum IL-1β levels from ApoE−/− mice were assessed by ELISA. Saline (n = 3), FSH-2w (n = 3), FSH-12w (n = 4). (H) The expression levels of CLDN12 in PMA/THP-1 cells were assessed by Western blot. Data shown in (A)–(F) and (H) are representative of three independent experiments. *p < 0.05, ** p < 0.01. ns, not significant.

FIGURE 4.

Effects of FSH on protein expression levels of inflammatory cytokines in macrophages. PMA/THP-1 cells were treated with FSH (50 ng/ml) for 24 h and the culture supernatant or cells were collected. (AC) The concentration levels of IL-1β (A), TNF-α (B), and IL-6 (C) in the culture supernatant of PMA/THP-1 cells were assessed by ELISA. (D) The expression levels of iNOS in PMA/THP-1 cells were assessed by Western blot. (E) The concentration levels of NO in the culture supernatant of PMA/THP-1 cells were assessed. (F) The expression levels of IL-1β in PMA/THP-1 cells were assessed by Western blot. (G) Serum IL-1β levels from ApoE−/− mice were assessed by ELISA. Saline (n = 3), FSH-2w (n = 3), FSH-12w (n = 4). (H) The expression levels of CLDN12 in PMA/THP-1 cells were assessed by Western blot. Data shown in (A)–(F) and (H) are representative of three independent experiments. *p < 0.05, ** p < 0.01. ns, not significant.

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NOS2 expresses iNOS, which catalyzes the formation of NO (30). NO is a biologically active free radical that acts in various physiological activities such as vasodilation and inflammation (31). Western blot detection showed that FSH did not clearly alter iNOS expression in macrophages (Fig. 4D). Further detection of the concentration of NO in the supernatant of the medium found that there was no significant difference between the FSH treatment and the control group (p = 0.3765) (Fig. 4E), indicating that FSH may not be involved in iNOS-related inflammatory responses.

Moreover, the serum IL-1β was detected in ApoE−/− mice with saline and with short- and long-term FSH exposure. Data from ELISAs showed, in ApoE−/− mice, that IL-1β was significantly increased with long-term FSH exposure and was slightly elevated with short-term FSH exposure (p = 0.0765) when compared with the vehicle control (Fig. 4G). These data suggested that FSH could contribute to AS development via enhancing the expression of IL-1β in macrophages.

Macrophage migration is a risk factor for many inflammatory diseases, autoimmune diseases, and cancer, and migration of macrophages to the site of inflammation due to recruitment of macrophages can lead to the accumulation of proinflammatory factors, tissue destruction, and the promotion of disease progression (32). AS is a chronic inflammatory cardiovascular disease, and the recruitment and accumulation of macrophages at the lesion site are crucial for its progression. Therefore, it was important to explore the effect of macrophage migration on the development of AS. In present study, when PMA/THP-1 cells were exposed to FSH, we found that FSH significantly promoted the migratory motility of macrophages (Fig. 5A). In AS patients, macrophages often engulf ox-LDL, resulting in the severe inhibition of migration ability (33). Therefore, we used ox-LDL as a migration inhibitor and found, after the phagocytosis of ox-LDL, that macrophage migration was barely affected, regardless of FSH treatment (Fig. 5A). These results suggested that FSH could significantly promote the migration of macrophages, but that it does not affect the migration of macrophages that have phagocytosed ox-LDL. Furthermore, the anti-FSHR Ab recovered the FSH-induced migration of macrophages (Fig. 5B). These data suggested that FSH may play a role in recruiting macrophages to the inflammatory sites before the phagocytosis of ox-LDL.

FIGURE 5.

FSH contributed to macrophage migration. (A) Effects of FSH with and without ox-LDL on macrophage migration were assessed by a Transwell assay. PMA/THP-1 cells were seeded on Transwell inserts (3 × 104 cells/insert) and treated with DMSO, FSH (50 ng/ml), ox-LDL and FSH+ox-LDL for 48 h. (B) Effects of FSH on macrophage migration with anti-IgG Ab or anti-FSHR Ab were assessed by a Transwell assay. With anti-IgG Ab or anti-FSHR Ab pretreatment, PMA/THP-1 cells were treated with FSH (50 ng/ml) for 24 h and then were seeded on Transwell inserts (3 × 104 cells/insert), and the lower chamber was filled with 20% FBS for 48 h. Scale bars, 20 μm. Data shown are representative of three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001. n.s., not significant.

FIGURE 5.

FSH contributed to macrophage migration. (A) Effects of FSH with and without ox-LDL on macrophage migration were assessed by a Transwell assay. PMA/THP-1 cells were seeded on Transwell inserts (3 × 104 cells/insert) and treated with DMSO, FSH (50 ng/ml), ox-LDL and FSH+ox-LDL for 48 h. (B) Effects of FSH on macrophage migration with anti-IgG Ab or anti-FSHR Ab were assessed by a Transwell assay. With anti-IgG Ab or anti-FSHR Ab pretreatment, PMA/THP-1 cells were treated with FSH (50 ng/ml) for 24 h and then were seeded on Transwell inserts (3 × 104 cells/insert), and the lower chamber was filled with 20% FBS for 48 h. Scale bars, 20 μm. Data shown are representative of three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001. n.s., not significant.

Close modal

Macrophages differentiate into foam cells after the phagocytosis of ox-LDL, and they then undergo apoptosis and accumulate to become part of AS plaques (26). Therefore, we tested the effect of FSH on the phagocytosis of ox-LDL by macrophages and found that the size of lipid droplets in macrophages with FSH exposure was not significantly different from that in the vehicle control, indicating that the ability of macrophages to take up lipids was not significantly altered by FSH stimulation (Supplemental Fig. 2C), suggesting that FSH had few effects on macrophage ox-LDL uptake.

Otherwise, macrophages were considered terminally differentiated cells and basically had no or weak proliferation ability. However, recent studies have demonstrated that macrophages can proliferate under specific conditions (34). With regard to this finding, we explored whether FSH regulates the proliferation of macrophages. PMA/THP-1 cells were treated with gradient concentrations of FSH for 24, 48, and 72 h and then CCK-8 was used to detect the proliferation. The results showed, at 24, 48, and 72 h, different concentrations of FSH had no clear effect on cellular proliferation compared with vehicle control (Supplemental Fig. 2D), meaning that FSH could barely stimulate macrophage proliferation.

Drug castration therapy is one of the most important treatment methods for prostate cancer, which can achieve good tumor control, but the cardiovascular complications have become the first cause of death in this type of patient. This was previously thought to be the result of decreased androgen levels, but clinical evidence suggested that other factors were involved. Therefore, clarifying pathogenesis is the key to effective intervention and prevention for this clinical problem.

FSH and its receptor (FSHR) play important roles in physiology. The conventional view holds that FSH has little relevance to the immune system. At present, there is a lack of research on the role of non-gonad–derived FSH on immune cells. Recently, several studies have revealed that FSH receptors are expressed in monocytes/macrophages and T cells; that is, these FSHR-expressing cells may be the potential targets of FSH. This study focused on the new physiological and pathological effects of FSH and explored whether FSH affects macrophages, which are related to AS, thereby providing new insights for research on cardiovascular diseases and other chronic inflammatory diseases.

In this study, we found that FSH may be the key factor to activate macrophages via improving the secretion of inflammatory factor IL-1β and promoting migration in ADT-treated patients with prostate cancer. In animal experiments, although long-term high levels of FSH induced elevated cholesterol in mice, short-term FSH did not, and both short-term and long-term high FSH could lead to similar atherosclerotic lesions, suggesting that the aggravation of AS by FSH was not primarily achieved by regulating cholesterol.

In in vitro studies, we found that FSH could activate macrophages, increase the secretion of IL-1β, and promote the migration ability, together aggravating AS lesions. IL-1β plays a crucial role in the activation of endothelial cells by initiating new atherogenesis and inducing worse plaque stability (35). A large clinical trial, the Canakinumab Anti-Inflammatory Thrombosis Outcome Study (CANTOS), demonstrated the efficacy of anti-inflammatory therapy in the treatment of AS and highlighted the important role of IL-1β in promoting the development of AS (36). Compared with normal people, the mRNA level and protein expression of IL-1β in the epicardial adipose tissue of AS patients were significantly increased, which was positively correlated with AS severity (37). The present study revealed that FSH could stimulate macrophages to express and secrete IL-1β, resulting in an inflammatory response and suggesting that the abnormal high level of FSH contributes to AS via activating macrophage inflammation. However, FSH could not promote the similar regulation of IL-1β in M1 macrophages, indicating that FSH could merely induce the initial differentiation of the inflammatory phenotype of macrophages, which would contribute inflammation. In experimental AS, selective neutralization of IL-1β has been shown to promote secretion of the anti-inflammatory factor IL-10 and to reduce AS plaque size (38). Therefore, the specific mechanism of FSH-induced IL-1β expression in macrophages deserves further exploration. Several studies have reported that activation of the NLRP3 inflammasome in monocyte/macrophage species induces IL-1β secretion (39), which provided the direction for us to explore the mechanism of FSH regulation of macrophages next.

Macrophage migration is a risk factor for many inflammatory diseases, autoimmune diseases, and cancer. The migration of macrophages to sites of inflammation due to recruitment can lead to the accumulation of proinflammatory factors, tissue destruction, and promote disease progression (32). AS is a chronic inflammatory cardiovascular disease, and the recruitment and accumulation of macrophages at the lesion site is necessary for its progression. Therefore, it is of great significance to explore the effect of macrophage migration on the development of AS. Our exploration found that FSH clearly regulated the migration of macrophages, but not of the foam cells. This suggests that FSH may play a role in the initial recruitment of macrophages, resident in vascular tissues or derived from infiltered monocytes, to inflammatory sites, such as damaged endothelium, lipid streak, and plaques. When inflammatory macrophages engulf ox-LDL and differentiate into foam cells, trapped in vascular tissues.

Until now, formal guidelines specifically for the prevention and management of CVD in patients on ADT and menopausal women are still absent. Bhatia et al. (40) have adapted management for prostate cancer patients and developed a paradigm to control CV risk factors in cancer survivors, including increasing awareness of patients on the signs and symptoms of CVD, aspirin, controlling blood pressure, paying close attention to cholesterol, diet, and exercise, among which may also be beneficial to menopausal women. To avoid additional or novel safety issues such FSH escalation, the combination administration of a GnRH antagonist and GnRH agonist has been proposed as described by Garnick and Mottet (41). Otherwise, several explorations have proven through pharmacological strategies that inhibiting FSH action on its receptor or FSHβ subunit promotes health by reducing serum cholesterol (42), inhibiting fat accumulation, and stimulating new bone formation as well as reducing bone removal (4345). In conclusion, prostate cancer patients with cardiovascular risk factors who require ADT, as well as menopausal women, may benefit from the better control of FSH provided by FSHβ or FSHR blocking, providing an important insight for the research and development of new drugs.

We thank Yue Chen and Jing Wu at Medical and Health Analysis Center, Peking University, for useful analysis of the results from immunofluorescence staining. We also thank Wenting Li at the Department of Laboratory Animal Science, Peking University Health Science Center, for useful instructions of animal experiments.

This work was supported by Natural Science Foundation of China Grants 82071777 (to T.X.) and 31570938, 31771280, and 11732001 (to W.-j.Y.) and by Interdisciplinary Medicine Seed Fund of Peking University Grant BMU2020MX002 (to T.X.).

Conceptualization, T.X. and J.-l.H.; methodology, J.-l.H. and W.-j.Y.; bioinformatics analysis, Y.-x.S.; validation, J.-l.H.; formal analysis, J.-l.H.; investigation, J.-l.H. and T.X.; resources, T.X. and W.-j.Y.; data curation, J.-l.H.; writing—original draft preparation, J.-l.H.; writing—review and editing, T.X. and W.-j.Y.; visualization, J.-l.H. and Y.-x.S.; supervision, T.X., W.-j.Y., Y.D., and J.Z.; project administration, J.-l.H.; funding acquisition, T.X. and W.-j.Y. All authors have read and agreed to the published version of the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ADT

androgen deprivation therapy

ApoE−/−

apolipoprotein E–deficient

AS

atherosclerosis

CCK-8

Cell Counting Kit-8

CLDN12

claudin 12

CVD

cardiovascular disease

FSH

follicle-stimulating hormone

FSH-12w

12-wk treatment with FSH

FSH-2w

2-wk treatment with FSH

GnRH

gonadotropin-releasing hormone

iNOS

inducible NO synthase

ox-LDL

oxidized low-density lipoprotein

THPA

The Human Protein Atlas

TJP1

tight junction protein 1

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The authors have no financial conflicts of interest.

Supplementary data