Inhibition of TP signaling promotes endometriosis growth and neovascularization.

Endometriosis is highly dependent on angiogenesis and lymphangiogenesis. Prostaglandin E2, an arachidonic acid metabolite, has been shown to promote the formation of new blood and lymphatic vessels. However, the role of another arachidonic acid metabolite, thromboxane A2 (TXA2) in angiogenesis and lymphangiogenesis during endometriosis remains largely unexplored. Using a murine model of ectopic endometrial transplantation, fragments from the endometrium of WT donor mice were transplanted into the peritoneal walls of recipient WT mice (WT→WT), resulting in an increase in both the area and density of blood and lymphatic vessels. Upon transplantation of endometrial tissue from thromboxane prostanoid (TP) receptor (TXA2 receptor)­deficient (TP­/­) mice into TP­/­ mice (TP­/­â†’TP­/­), an increase in implant growth, angiogenesis, and lymphangiogenesis were observed along with upregulation of pro­angiogenic and lymphangiogenic factors, including vascular endothelial growth factors (VEGFs). Similar results were obtained using a thromboxane synthase (TXS) inhibitor in WT→WT mice. Furthermore, TP­/­â†’TP­/­ mice had a higher number of F4/80+ cells than that of WT→WT mice, with increased expression of genes related to the anti­inflammatory macrophage phenotype in endometrial lesions. In cultured bone marrow (BM)­derived macrophages, the levels of VEGF­A, VEGF­C, and VEGF­D decreased in a TP­dependent manner. Furthermore, TP signaling affected the polarization of cultured BM­derived macrophages to the anti­inflammatory phenotype. These findings imply that inhibition of TP signaling promotes endometrial implant growth and neovascularization.

Emotional memories are enhanced when reactivated in slow wave sleep, but impaired when reactivated in REM.

Sleep supports memory consolidation. However, it is not completely clear how different sleep stages contribute to this process. While rapid eye movement sleep (REM) has traditionally been implicated in the processing of emotionally charged material, recent studies indicate a role for slow wave sleep (SWS) in strengthening emotional memories. Here, to directly examine which sleep stage is primarily involved in emotional memory consolidation, we used targeted memory reactivation (TMR) in REM and SWS during a daytime nap. Contrary to our hypothesis, reactivation of emotional stimuli during REM led to impaired memory. Consistent with this, REM% was correlated with worse recall in the group that took a nap without TMR. Meanwhile, cueing benefit in SWS was strongly correlated with the product of times spent in REM and SWS (SWS-REM product), and reactivation significantly enhanced memory in those with high SWS-REM product. Surprisingly, SWS-REM product was associated with better memory for reactivated items and poorer memory for non-reactivated items, suggesting that sleep both preserved and eliminated emotional memories, depending on whether they were reactivated. Notably, the emotional valence of cued items modulated both sleep spindles and delta/theta power. Finally, we found that emotional memories benefited from TMR more than did neutral ones. Our results suggest that emotional memories decay during REM, unless they are reactivated during prior SWS. Furthermore, we show that active forgetting complements memory consolidation, and both take place across SWS and REM. In addition, our findings expand upon recent evidence indicating a link between sleep spindles and emotional processing.

Sphingosine 1-Phosphate (S1P) in the Peritoneal Fluid Skews M2 Macrophage and Contributes to the Development of Endometriosis.

Sphingosine 1-phosphate (S1P), an inflammatory mediator, is abundantly contained in red blood cells and platelets. We hypothesized that the S1P concentration in the peritoneal cavity would increase especially during the menstrual phase due to the reflux of menstrual blood, and investigated the S1P concentration in the human peritoneal fluid (PF) from 14 non-endometriosis and 19 endometriosis patients. Although the relatively small number of samples requires caution in interpreting the results, S1P concentration in the PF during the menstrual phase was predominantly increased compared to the non-menstrual phase, regardless of the presence or absence of endometriosis. During the non-menstrual phase, patients with endometriosis showed a significant increase in S1P concentration compared to controls. In vitro experiments using human intra-peritoneal macrophages (MΦ) showed that S1P stimulation biased them toward an M2MΦ-dominant condition and increased the expression of IL-6 and COX-2. An in vivo study showed that administration of S1P increased the size of the endometriotic-like lesion in a mouse model of endometriosis.

CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model.

In endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is unclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines and chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable to deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion of CD206+ MΦ decreased the total weight of endometriotic-like lesions significantly (p < 0.05). In the endometriotic-like lesions in the DT group, a lower proliferation of endometriotic cells and the decrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFß1, angiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, respectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell marker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number of TGFß1 expressing MΦ was significantly reduced compared to control. These data suggest that CD206+ MΦ promotes the formation of endometriotic-like lesions by inducing angiogenesis around the lesions.

CD206+ M2-Like Macrophages Are Essential for Successful Implantation.

Macrophages (MΦs) play important roles in implantation. Depletion of CD11b+ pan-MΦs in CD11b-diphtheria-toxin-receptor (DTR) mice is reported to cause implantation failure due to decreased progesterone production in the corpus luteum. However, of the M1 and M2, the type of MΦs that is important for implantation is unknown. In this study, we investigated the role of M2 MΦ in implantation using CD206-DTR mice. To deplete M2-MΦ, female CD206-DTR C57/BL6 mice were injected with DT before implantation. These M2-MΦ depleted mice (M2(-)) were naturally mated with Balb/C mice. As the control group, female C57/BL6 wild type (WT) mice injected with DT were mated with male Balb/C mice. The number of implantation sites and plasma progesterone levels at implantation were examined. Implantation-related molecule expression was determined using quantitative-PCR and immunohistochemistry of uterine tissues. The mRNA expression in the endometrial tissues of 38 patients with implantation failure was examined during the implantation window. In WT mice, CD206+M2-like MΦs accumulated in the endometrium at the implantation period, on embryonic (E) 4.5. In M2(-), the implantation number was significantly lower than that in control (p < 0.001, 7.8 ± 0.8 vs. 0.2 ± 0.4), although the plasma progesterone levels were not changed. Leukemia inhibitory factor (LIF) and CD206 mRNA expression was significantly reduced (p < 0.01), whereas the levels of TNFα were increased on E4.5 (p < 0.05). In M2(-), the number of Ki-67+ epithelial cells was higher than that in control at the pre-implantation period. Accelerated epithelial cell proliferation was confirmed by significantly upregulated uterine fibroblast growth factor (FGF)18 mRNA (P < 0.05), and strong FGF18 protein expression in M2(-) endometrial epithelial cells. Further, M2(-) showed upregulated uterine Wnt/ß-catenin signals at the mRNA and protein levels. In the non-pregnant group, the proportion of M2-like MΦ to pan MΦ, CD206/CD68, was significantly reduced (p < 0.05) and the TNFα mRNA expression was significantly increased (p < 0.05) in the endometrial tissues compared to those in the pregnant group. CD206+ M2-like MΦs may be essential for embryo implantation through the regulation of endometrial proliferation via Wnt/ß-catenin signaling.

Relaxin-2 May Suppress Endometriosis by Reducing Fibrosis, Scar Formation, and Inflammation.

BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.

IL-33 Exacerbates Endometriotic Lesions via Polarizing Peritoneal Macrophages to M2 Subtype.

In endometriosis, M2 macrophages (MΦ) are dominant and promote the development of endometriosis lesions. However, the factor(s) which induces M2 MΦ are unknown. In the present study, we focused on interleukin (IL)-33, known as an alarmin and investigated its expression and its role in endometriosis, especially from the point of the relevance with MΦ. The expression of IL-33 in endometriosis lesions was examined by immunohistochemistry. The cystic fluid of ovarian cysts/tumors was obtained and used to measure IL-33 concentration. Endometriotic stromal cells (ESC) and MΦ derived from patients were used for in vitro experiments. IL-33 was detected in the epithelium and stromal cells of endometriotic lesions. The mean IL-33 concentration in the cystic fluid of endometriomas was significantly higher than that in non-endometriomas (2.2 ng/ml vs. 0.02 ng/ml, P < 0.01). IL-1ß induced IL-33 mRNA expression in ESC via p38 MAPK activation. With IL-33 stimulation, peritoneal MΦ polarized to M2 MΦ and produced IL-1ß mRNA with a 2.2-fold increase, which was negated with soluble ST2, a decoy receptor of IL-33. IL-33, derived from endometriotic lesions, stimulated MΦ to produce IL-1ß, which results in increasing IL-33 production in ESC. This cycle may continue to exacerbate the endometriotic lesions.

Topographic distribution of lymphatic vessels in the normal human prostate.

BACKGROUND: The lymphatic endothelial hyaluronan receptor (LYVE-1) is a specific cell surface protein in lymphatic endothelium. The antiserum against human LYVE-1 was developed and was confirmed a powerful marker of lymphatic endothelium in human organs. With this novel marker we investigated the small network of intraprostate lymphatic vessels. METHODS: To identify intraprostatic lymphatic vessels, we performed an immunohistochemical staining method using LYVE-1 pAb and von Willebrand Factor (vWF), and assessed the difference in distribution of small lymphatic vessels between the components in the prostate. RESULTS: The density of lymphatic vessel was significantly high around ejaculatory ducts and in the fibromascular area between the globular area of peripheral zone and transitional zone. Predominat lymphatic vessels distributed in the fibromascular area in the anterior and posterior prostate of extrastromal area. CONCLUSIONS: Recognition of the distinctive features of the intraprostate lymphatic network, can help the investigation of lymphatic involvement in cancer of the prostate.