Control Endometrial Stromal Cells Research Articles

Effect of active vitamin D on proliferation, cell cycle and apoptosis in endometriotic stromal cells

Research questionWhat is the effect of vitamin D3 (1,25(OH)2D3) on proliferation, cell cycle and apoptosis of endometrial stromal cells (ESC) in endometriotic patients? DesignESC isolated from 10 women with endometriosis and 10 healthy controls were treated with 1,25(OH)2D3. The proliferation of control endometrial stromal cells (CESC), eutopic endometrial stromal cells (EuESC) and ectopic endometrial stromal cells (EESC) was analysed 72 h after the treatment using methyl thiazolyl tetrazolium assay. Propidium iodide staining and flow cytometry were used to determine the cell cycle distribution in ESC. Annexin V/propidium iodide double staining was used to evaluate apoptosis in ESC. ResultsIn the presence of oestrogen, 1,25(OH)2D3 treatment inhibited the proliferation of ESC from all three origins (P = 0.009 for CESC, P = 0.005 for EuESC and P < 0.001 for EESC). The percentage of S phase cells in EESC was higher than in EuESC and CESC (P = 0.002 and P = 0.001, respectively). The percentage of S phase cells in EuESC was higher than in CESC (P = 0.005). The percentage of G1 phase cells in EESC was lower than that of EuESC and CESC (P = 0.003 and P = 0.002, respectively) and the percentage of G1 phase cells in EuESC was lower than that of CESC (P = 0.007). Moreover, 1,25(OH)2D3 inhibited cell cycle regardless of cell type (P = 0.002 in EESC, P = 0.001 in EuESC and P = 0.014 in CESC), but in the absence of oestrogen, inhibited cell cycle only in EuESC (P = 0.012). ConclusionsAlthough 1,25(OH)2D3 increased apoptotic and necrotic cells and decreased live cells in the EuESC and EESC, it did not affect apoptosis in CESC and only increased necrotic cells. These findings indicate that 1,25(OH)2D3 potentially has a growth-inhibiting and pro-apoptotic effect on ESC from endometriotic patients.

P–285 Controlling semi-invasive activity of human endometriotic stromal cells by inhibiting NF-kB signaling pathway using aloe-emodin or aspirin

Abstract Study question Does inhibiting nuclear factor-kB (NF-kB) signaling by aloe-emodin (AE) or aspirin (Asp), as anti-inflammatory compounds, suppress the invasive activity of stage IV human endometriotic stromal cells? Summary answer Eutopic endometriotic stromal cells (EuESCs) seem to have a semi-invasive activity which is largely suppressed by AE or Asp. What is known already Inflammation and its master regulator, NF-kB, have been implicated in the development of endometriosis. Inhibition of NF-kB pathway using small molecules ameliorated disease progression and reduced the lesion size; nevertheless, underlying mechanism is not fully understood. Study design, size, duration In this cross-sectional study, a total of 8 infertile patients with proven endometriosis and 8 women without endometriosis (Control group) undergoing infertility treatment cycles, were enrolled between October 2018 and December 2019. The invasiveness of collected endometriotic stromal cells before and after treatment with AE or Asp, was analyzed and compared with the control group. Participants/materials, setting, methods The eutopic endometriotic and healthy endometrial biopsies were digested and the single cells were cultured. Gene and protein expression of proliferation, adhesion, and invasion markers of eutopic endometriotic stromal cells (EuESCs) with and without treatment with AE or Asp, as well as control endometrial stromal cells (CESCs) were analyzed using q-PCR and immunofluorescence staining, respectively. Cell migration capacity was assessed by wound closure assay. Main results and the role of chance We observed an association between NF-kB overexpression and higher proliferation/adhesion capacity in EuESCs. TNF-α, as a known NF-kB inducer, further potentiated this association. EuESCs at stage IV, displayed silent invasive and migratory behaviors. Pretreatment of EuESCs with AE or Asp significantly attenuated NF-kB expression and reduced proliferative, adhesive, invasive and migratory activity. Limitations, reasons for caution Due to some adverse effects observed following treatment with AE or Asp on the normal activity of EuESCs, more investigations on possible toxicity of the treatment, must be considered. Wider implications of the findings: We suggest that both Asp and AE (as potent NF-kB inhibitors) may be useful as a supplement to conventional endometriosis treatments. Trial registration number Not applicable

The effects of resveratrol on the expression of VEGF, TGF-\u03b2, and MMP-9 in endometrial stromal cells of women with endometriosis

Resveratrol is a phytochemical with anti-angiogenic, anti-inflammatory, and antioxidant properties. The present study has evaluated the effect of resveratrol on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloproteinase-9 (MMP-9) as factors related to endometriosis progression. Thirteen eutopic (EuESCs) and 8 ectopic (EESCs) endometrial stromal cells from women with endometriosis and 11 control endometrial stromal cells (CESCs) were treated with resveratrol (100 µM) for 6, 24 and 48 h. The gene and protein expression levels of VEGF, TGF-β, and MMP-9 were measured using real-time PCR and ELISA methods, respectively. Results showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs compared to EuESCs and CESCs (P < 0.01 to < 0.001 and P < 0.05 to < 0.01 respectively). Also, resveratrol treatment decreased the gene and protein expression of VEGF and MMP-9 in EuESCs, EESCs and CESCs (P < 0.05 to < 0.01 and P < 0.05 to < 0.01 respectively) and gene and protein expression of TGF-β in EESCs and EuESCs (P < 0.05 to < 0.01). The effect of resveratrol in reduction of VEGF gene expression was statistically more noticeable in EESCs compared to EuESCs and CESCs (P < 0.05). According to the findings, resveratrol may ameliorate endometriosis progression through reducing the expression of VEGF, TGF-β, and MMP-9 in endometrial stromal cells (ESCs).

Controlling Semi-Invasive Activity of Human Endometrial Stromal Cells by Inhibiting NF-kB Signaling Pathway Using Aloe-emodin and Aspirin

Background:Inflammation and its master regulator, Nuclear Factor-kB (NF-kB), have been implicated in the development of endometriosis. Inhibition of NF-kB pathway using small molecules ameliorated disease progression and reduced the lesion size; nevertheless, the underlying mechanism is not fully understood. Therefore, this study, is an attempt to assess whether inhibiting NF-kB signaling by aloe-emodin (AE) or aspirin (Asp), as anti-inflammatory compounds, can suppresses the invasive activity of human endometrial stromal cells at stage IV endometriosis.Methods:The eutopic and healthy endometrial biopsies from a total of 8 infertile women with confirmed endometriosis and 8 women without endometriosis were digested and the single cells were cultured. Gene and protein markers of proliferation, migration, adhesion, and invasion of eutopic endometrial stromal cells (EuESCs) with and without treatment with AE or Asp, as well as control endometrial stromal cells (CESCs) was analyzed using q-PCR and immunofluorescence staining, respectively. Comparison between groups was performed using one-way ANOVA and the Bonferroni post hoc and p≤0.5 was considered statistically significant.Results:There was an association between NF-kB overexpression and higher proliferation/adhesion capacity in EuESCs. EuESCs (at stage IV endometriosis) displayed no invasive and migratory behaviors. Pre-treatment of EuESCs with AE or Asp significantly attenuated NF-kB expression and reduced proliferative, adhesive, invasive, and migratory activity of endometrial cells (p≤0.5).Conclusion:Eutopic endometrial stromal cells seem to have a semi-invasive activity which is largely suppressed by AE or Asp. It can be suggested that both Asp and AE (as potent NF-kB inhibitors) can be used as a supplement in conventional endometriosis treatments.

Emodin Reverses the Epithelial-Mesenchymal Transition of Human Endometrial Stromal Cells by Inhibiting ILK/GSK-3β Pathway.

PurposeTo explore the exact mechanism through which emodin down-regulates the migration and invasion abilities of endometrial stromal cells. Moreover, to explore the theoretical basis of emodin in the treatment of endometriosis.Patients and MethodsEndometriosis endometrial stromal cells (EESs) were cultured from 15 women with endometriosis and control endometrial stromal cells (CESs) were cultured from 12 women without endometriosis. The levels of proteins were evaluated by Western blot. The migration and invasion abilities of cells were detected by transwell assays.ResultsThe abilities of migration and invasion of EESs were much stronger than those of CESs. After treated with emodin, the migration and invasion abilities of EESs and CESs were significantly down-regulated, and the levels of integrin-linked kinase (ILK) and p-GSK-3β were statistically down-regulated in EESs. Besides that, the expression of keratin was up-regulated while the expression of vimentin, β-catenin and slug were all down-regulated by emodin in a dose- and time-dependent manner. Silencing of ILK gene in EESs also achieved the above effects, which were strengthened by emodin. Conversely, exogenous expression of ILK in CESs increased the expression of p-GSK-3β, which were abrogated by emodin. Furthermore, SB216763 increased migration and invasion abilities of CESs by facilitating the epithelial–mesenchymal transition (EMT) through up-regulating levels of p-GSK-3β, β-catenin and slug, which were also abrogated by emodin.ConclusionEmodin inhibits the migration and invasion abilities of human endometrial stromal cells by reversing the EMT via ILK/GSK-3β pathway. So, emodin may be considered as a promising targeted therapy for endometriosis.

MiR-199a-5p Targets ZEB1 to Inhibit the Epithelial-Mesenchymal Transition of Ovarian Ectopic Endometrial Stromal Cells Via PI3K/Akt/mTOR Signal Pathway In Vitro and In Vivo.

Endometriosis (Ems) is a common gynecological disease with the characteristics of infertility, pelvic pain, and sexual intercourse difficulty. Our present study aimed to investigate the effect of miR-199a-5p on cell mobility and epithelial-mesenchymal transition (EMT) in Ems. Ectopic endometrial stromal cells (EcSCs) and control endometrial stromal cells (CSCs) were isolated in our in vitro experiments. The level of miR-199a-5p in EcSCs was found much lower than that in CSCs. Besides, miR-199a-5p mimic suppressed the invasion and migration ability of EcSCs. At the same time, EMT was also found to be suppressed by miR-199a-5p mimic in EcSCs. Our further bioinformatics analysis and luciferase reporter assay revealed that ZEB1, a marker of EMT, was a direct target of miR-199a-5p. In addition, the combination of pcDNA3.1-ZEB1 weakened the inhibiting effect of miR-199a-5p mimic on the mobility and EMT of EcSCs. What is more, the PI3K/Akt/mTOR signal pathway was demonstrated to be inactivated by miR-199a-5p mimic. And then, the inducer of PI3K/Akt/mTOR signal pathway, IGF-1, abolished the effect of miR-199a-5p mimic on Ems progression. At last, an Ems rat model was established, and we found that miR-199a-5p agomir effectively suppressed the expression of vascular endothelial growth factor (VEGF) and EMT in vivo. The PI3K/Akt/mTOR signal pathway was also inactivated by miR-199a-5p agomir in our Ems rat model. Taken together, we concluded that miR-199a-5p targeted ZEB1 to inhibit the EMT of ovarian ectopic endometrial stromal cells via PI3K/Akt/mTOR signal pathway in vitro and in vivo, advancing our understanding of miR-199a-5p as regulators of Ems progression and making contribution to the treatment of Ems.

Resveratrol reduces the expression of insulin-like growth factor-1 and hepatocyte growth factor in stromal cells of women with endometriosis compared with nonendometriotic women.

Resveratrol, a phytoalexin polyphenol, has antiproliferative, antiangiogenic, anti-inflammatory, and antioxidant properties. The present study has assessed the effect of resveratrol treatment on the expression of insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) in endometrial stromal cells (ESCs) from women with and without endometriosis. Endometrial tissues were obtained from 40 endometriotic patients and 15 nonendometriotic control women. After the enzymatic digestion, 13 eutopic ESCs (EuESCs), 8 ectopic ESCs (EESCs), and 11 control ESCs (CESCs) were treated with resveratrol (100μM) for 6, 24, and 48hr. The gene and protein expressions of IGF-1 and HGF were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. Results showed that resveratrol treatment decreased significantly the gene expression of IGF-1 and HGF in EuESCs, EESCs, and CESCs (p<0.05). The effect of resveratrol treatment on the reduction of IGF-1 gene expression was statistically more noticeable in EESCs compared with CESCs (p<0.05). Also, in the case of HGF gene expression, the reducing effect of resveratrol treatment was statistically more considerable in EESCs compared with EuESCs and CESCs (p<0.05 and p<0.01, respectively). The IGF-1 and HGF protein production decreased significantly in EuESCs and EESCs (p<0.05) but not in CESCs. These findings suggest that resveratrol treatment could reduce the expression of IGF-1 and HGF in ESCs especially in EESCs, which play a pivotal role in disease progression.

ILK enhances migration and invasion abilities of human endometrial stromal cells by facilitating the epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT) plays a significant part in the pathogenesis of endometriosis by facilitating the migration and invasion abilities of cells. Integrin-linked kinase (ILK) increases the cell migration and invasion abilities by inducing the EMT. Eutopic and control endometrial stromal cells (EuSCs and CSCs) were isolated and cultured. Cell migration and invasion abilities were detected by transwell assays. Levels of proteins were detected by Western blot. EuSCs showed higher levels of ILK, N-cadherin, vimentin and stronger migration and invasion abilities. After transfection of siRNA-ILK, E-cadherin and keratin levels were increased while N-cadherin and vimentin levels were decreased in EuSCs. Besides that, the migration and invasion abilities of EuSCs were significantly decreased after transfection of siRNA-ILK. On the contrary, levels of ILK, N-cadherin and vimentin were increased while levels of E-cadherin and keratin were decreased simultaneously after transfecting CSCs with pEGFP-C1-ILK. Simultaneously, the migration and invasion abilities of CSCs were increased after transfection of pEGFP-C1-ILK. Our study verified that high expression of ILK enhanced the migration and invasion abilities of ESCs by facilitating the EMT. Given that ILK played crucial roles in the pathogenesis of endometriosis, it may be considered as a promising targeted therapy for endometriosis.

Emodin Inhibits Migration and Invasion of Human Endometrial Stromal Cells by Facilitating the Mesenchymal-Epithelial Transition Through Targeting ILK.

To determine whether emodin facilitates the mesenchymal-epithelial transition (MET) of endometrial stromal cells (ESCs) as well as to explore the mechanism through which emodin favored the MET of ESCs. Cell viability was tested by methyl thiazolyl tetrazolium assay. Cell migration and invasion abilities were detected by transwell assays. Levels of integrin-linked kinase (ILK) and epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. Upregulated ILK and increased abilities of migration and invasion were confirmed in the eutopic and ectopic ESCs (EuSCs and EcSCs), especially in the EcSCs. After treated with emodin, the expression of ILK was statistically downregulated in EcSCs, resulting in the MET and decreased migration and invasion abilities of EcSCs. Additionally, silencing of the ILK gene in EcSCs also achieved the above-mentioned effects, which were strengthened by emodin. Furthermore, exogenous expression of ILK in control ESCs (CSCs) resulted in the EMT and increased abilities of migration and invasion of CSCs, which can be abrogated by emodin. Besides, exogenous expression of ILK also abrogated the effects of emodin on CSCs. Emodin inhibits the migration and invasion abilities of human ESCs by facilitating the MET through targeting ILK.

Effect of progesterone on renin secretion in endometrial stromal, chorionic trophoblast, and mesenchymal monolayer cultures

Renin messenger ribonucleic acid expression has been shown in decidual tissue and endometrium, but weakly in chorion laeve, suggesting decidua as a primary source of uteroplacental renin. We proposed to confirm active renin secretion by endometrial stromal cells and examined the effect of progesterone-induced decidualization on secretion. Separate monolayer cultures of endometrial stroma, trophoblast, and mesenchymal cells were established and maintained for 10 to 15 days. Active renin concentration was measured with radioimmunoassay for angiotensin I generation and expressed as picograms per milliliter of angiotensin I generation per 10(5) cells. Active renin concentration was high in control secretory endometrial stromal cells (513 pg/ml) compared with trophoblast (none) and mesenchymal cell cultures (74 pg/ml). Marked decrease in active renin secretion occurred in control endometrial stromal cells (days 13 to 15, 86 pg/ml), whereas progesterone-induced decidualization sustained and increased secretion (days 13 to 15, 1017 pg/ml). This renin activity was quantitatively inhibited in culture fluid assays by specific human renal renin antibody in serial dilutions. Renin activity measurements before and after trypsin activation showed the majority of renin (91.15%) in the inactive form and a smaller fraction (8.85%) in the active form. Immunohistochemistry with the use of specific human renal renin antibody confirmed the presence of renin and its stimulation by progesterone in endometrial stromal cells. Progesterone had minimal effect on mesenchymal cells. These data confirmed endometrial stromal cells as a significant source of active renin and showed that progesterone induced a marked increase in this production.