Human Endometrial Endothelial Cells Research Articles

PGE2 Regulates the Plasminogen Activator Pathway in Human Endometrial Endothelial Cells: A New In Vitro Model to Investigate Heavy Menstrual Bleeding

To study the role of PGE2 in regulating plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in human primary endometrial endothelial cells (HEECs) from women with normal menstrual bleeding (NMB) and heavy menstrual bleeding (HMB). In vitro study using endometrial endothelial cells. Research laboratory setting. Women with normal menstrual bleeding (NMB) and heavy menstrual bleeding (HMB) provided endometrial biopsy samples. PGE2 and PGE2 receptor-selective agonists were administered to cultured HEECs. Levels of PAI-1 and tPA in NMB-HEECs and HMB-HEECs after treatment with PGE2 and receptor-selective agonists. PGE2 increased total PAI-1 levels in NMB-HEECs, but not in HMB-HEECs, which had higher baseline PAI-1 levels. PTGER1 and PTGER2 agonists increased PAI-1 in NMB-HEECs, while PTGER3 and PTGER4 did not. PGE2 had no effect on tPA levels in either NMB-HEECs or HMB-HEECs. PGE2, through PTGER1 and PTGER2, regulates the plasminogen activator system in NMB-HEECs, suggesting a role in reducing fibrinolytic activity during normal menstrual cycles. The lack of PGE2 effect and elevated baseline PAI-1 in HMB-HEECs support using this in vitro model to further understand prostaglandin pathways in normal and heavy menstrual bleeding.

Antiphospholipid-exposed trophoblast-derived extracellular vesicles express elevated levels of TLR7/8-activating microRNAs and induce endometrial endothelial activation, in part, through TLR7

Women with antiphospholipid syndrome (APS) are at high risk for miscarriage and preeclampsia. Unlike pro-thrombotic systemic APS, obstetric APS is associated with insufficient placentation, as well as inflammation and vascular dysfunction at the maternal-fetal interface. Antiphospholipid antibodies (aPL) can target the placental trophoblast and induce inflammation. We reported that aPL trigger trophoblast cells to produce elevated levels of IL-8 through activation of Toll-like receptor 4 (TLR4). Downstream of TLR4, we found this IL-8 response is mediated by a TLR8-activating microRNA (miR), miR-146a-3p, which is also released by the trophoblast via extracellular vesicles (EVs). Since endothelial dysfunction is a feature of obstetric APS, we sought to determine if other miRs that can activate the RNA sensors, TLR7 and/or TLR8, are released by the trophoblast via EVs after exposure to aPL, and if these EVs can activate human endometrial endothelial cells (HEECs). Using a human first trimester extravillous trophoblast cell line we found that aPL elevated their release of small EVs (<150 nm). These extracellular vesicles released from trophoblast cells exposed to aPL expressed elevated levels of TLR7/8-activating miR-21a and miR-29a, in addition to the previously reported miR-146a-3p. Extracellular vesicles from aPL-exposed human trophoblast cells triggered human endometrial endothelial cells to generate an inflammatory IL-8 response, in part through TLR7. This study highlights EVs as a mode of communication between the placenta and the maternal vasculature, as well as a potential role for TLR7/8-activating miRs in contributing to inflammation at the maternal-fetal interface in obstetric APS.

PGE2 REGULATES THE PLASMINOGEN ACTIVATOR PATHWAY IN HUMAN ENDOMETRIAL ENDOTHELIAL CELLS THROUGH PROSTAGLANDIN-E2 RECEPTORS (PTGER) 1 AND 2

This study seeks to determine which prostaglandin E2 receptor (PTGER) mediates alterations in plasminogen activator inhibitor-1 (PAI-1) levels in human endometrial endothelial cells (HEEC).

GCN5 participates in KLF4-VEGFA feedback to promote endometrial angiogenesis

SummaryEndometrial angiogenesis is necessary for good endometrial receptivity. Krüppel-like factor 4 (KLF4) is a transcription factor that is essential for regulating angiogenesis. Here we found that vascular endothelial growth factor A (VEGFA) can form a positive feedback loop with KLF4 to promote the proliferation and migration of human endometrial microvascular endothelial cells (HEMECs) and inhibit cell apoptosis. General control non-derepressible 5 (GCN5) is also time-dependent on VEGFA and participates in the KLF4-VEGFA loop. In addition, we found that GCN5 is a succinyltransferase that modulates the succinylation of histones and nonhistones. GCN5 interacts with KLF4 and is recruited to the KLF4-binding site of the VEGFA promoter to succinylate H3K79, which initiates gene transcription epigenetically. For nonhistones, GCN5 succinylates KLF4 that is activated by ERK signaling in HEMECs treated with VEGFA to increase its transcription activity. These results demonstrate KLF4-VEGFA positive feedback loop is regulated by epigenetics, which contributes to endometrial angiogenesis.

Report of Exosomes Isolated from a Human Uterine Leiomyoma Cell Line and Their Impact on Endometrial Vascular Endothelial Cells.

Uterine leiomyomas are the most common pelvic tumor in women of reproductive age; they cause irregular heavy menstrual bleeding leading to anemia and subsequent negative effects on quality of life. Exosomes have arisen as main players of disease progression in several illnesses, including a range of benign and malignant conditions; however, their role in leiomyomas’ pathophysiology remains unknown. We investigated the effect of exosomes derived from human uterine leiomyoma tumor cells (HULM) and human myometrial cells (UTSM) on the behavior of human endometrial microvascular endothelial cells (HEMEC). HULM- and UTSM-derived exosomes were isolated and cocultured with HEMECs. Then, cell proliferation, mRNA expression, tube formation assay, and RNA-seq were performed. Treatment of HEMEC with HULM-derived exosomes increased cell proliferation by 60% compared to control untreated cells, upregulated C-MYC and VEGFA expression levels, and increased tube formation, length, and branching (markers of angiogenesis). Profiling of miRNA revealed that 84 miRNAs were significantly downregulated and 71 were upregulated in HULM-derived exosomes compared to UTSM-derived exosomes. These findings suggest that HULM-derived exosomes might have effects on HEMEC function, containing factors that enhance endometrial proliferation and angiogenesis, which may contribute to heavy menstrual bleeding. Further research on exosomes in uterine leiomyoma may identify possible novel biomarkers for treatment.

PGE2 REGULATES THE PLASMINOGEN ACTIVATOR PATHWAY IN HUMAN ENDOMETRIAL ENDOTHELIAL CELLS: DIFFERENCES BETWEEN NORMAL AND HEAVY MENSTRUAL BLEEDERS

This study seeks to determine if Prostaglandin E2 (PGE2) alters plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in human endometrial endothelial cells (HEEC) and identify any differences between women with normal and heavy menstrual bleeding. HEEC primary cultures were established from two groups of women: heavy menstrual bleeding (HMB, n=2), and normal menstrual bleeding (NMB, n=3). HEEC were cultured with PGE2 at concentrations of 10-7 10-9, 10-11 M and no PGE2 as a control. We used ELISAs to assess the concentration of PAI-1 and tPA in media; total protein was extracted from the cells and determined using the Bicinchoninic Acid protein assay. There were no differences in patient demographics, the average age in NMB 27.7 and HMB was 31.2 (p=0.44). HMB women had at least one fibroid by ultrasound. There was an increase in PAI-1 concentration in HEEC exposed to both 10-7 and 10-9 M of PGE2 in NMB, 332 ±183 and 248-±70 % of control, respectively. HMB women had a different outcome with PAI-1 where concentrations decreased 68 ±29 and 59-±22 % of control at both 10-7 and 10-9 M of PGE2, respectively. There was no change in tPA concentration 111 ±43 and 103±19% of control in NMB at 10-7 and 10-9 M of PGE2, respectively, but a decrease in tPA concentration when PGE2 was added to the cell cultures of HMB women, 68 ±4, and 64 ±16 % control with 10-9 and 10-11 M of PGE2 respectively (Table 1). PGE2 increased PAI-1 concentration in endothelial cells of NMB, which would lead to more stable clots during menses and an objectively lighter menses. PAI-1 secretion from endothelial cells of HMB women did not respond. This suggests that the endothelium of HMB women may be dysregulated in their PAI-1/tPA pathway in response to PGE2, which may be a central cause of their heavier menses.

Intentional endometrial injury enhances angiogenesis through increased production and activation of MMP-9 by TNF-α and MMP-3 in a mouse model.

There have been reports of improved pregnancy rates after performing intentional endometrial injuries, also known as endometrial scratching, in patients with recurrent implantation failure. In our previous study on intentional endometrial injury, we found an increased expression of matrix metalloproteinase (MMP)-3 following induced injuries to the mice endometrium. In the current study, we further examine whether the rise in MMP-3 could contribute to increased angiogenesis. Female C57B1/6 mice were obtained at 12 weeks of age, and intentional endometrial injuries were induced mechanically in the left uterine horns. Using the appropriate media, uterine-washes were performed on the injured and uninjured (control) horns of the harvested uteri. The uterine tissues were further processed for tissue lysates, histopathology and immunohistochemistry. The results show that intentional endometrial injuries caused an increase in secreted LPA in the injured horns, which were detected in the uterine-washes. In addition, LPA induced increased production of TNF-α in human endometrial epithelial cells (hEEpCs). Furthermore, TNF-α appeared to induce differential and cell-specific upregulation of the MMPs: MMP-3 was upregulated in the epithelial (hEEpCs), while MMP-9 was upregulated in the endothelial cells (human endometrial endothelial cells; hEEnCs). The upregulation of MMP-3 appeared to be necessary for the activation of MMP-9, whose active form stimulated the formation of vessel-like structure by the hEEnCs. The results of this study suggest that there may be enhanced angiogenesis following intentional endometrial injuries, which is mediated in part by TNF-α-induced and MMP-3-activated MMP-9 production.

SIRT1-induced deacetylation of Akt expedites platelet phagocytosis and delays HEMEC aging

Maintaining the health of the endothelium is of critical importance to prevention against cell aging. The current study was performed to clarify the role of sirtuin1 (SIRT1) in platelet phagocytosis in cell aging and identified its downstream molecular mechanism. Platelet phagocytosis by human endometrial microvascular endothelial cells (HEMECs) was characterized by transmission electron and fluorescence microscopy. Functional experiments were conducted to examine platelet phagocytosis and cell aging using the overexpression or knockdown plasmids of SIRT1 and G alpha-interacting, vesicle-associated protein (GIRDIN) as well as Akt inhibitor and activator. It was found that SIRT1 facilitated platelet phagocytosis by HEMECs, contributing to inhibition of cell aging. Akt activation facilitated platelet phagocytosis and repressed cell aging. GIRDIN overexpression accelerated platelet phagocytosis by HEMECs, leading to a delay in cell aging. GIRDIN phosphorylation at Ser1417 was induced by Akt activation, while activation of Akt was induced by SIRT1-mediated deacetylation, consequently augmenting platelet phagocytosis and delaying cell aging. Taken together, SIRT1 delayed aging of HEMECs by deacetylating Akt, phosphorylating GIRDIN, and inducing platelet phagocytosis. The study highlights a possible target for the prevention of HEMEC aging.

Estrogen Regulates Endoplasmic Reticulum Stress-Mediated Apoptosis by ERK-p65 Pathway to Promote Endometrial Angiogenesis.

Estrogen (17β-oestradiol, E2) plays an essential role in endometrial receptivity and has been shown to stimulate angiogenesis via E2-ERα (estrogen receptor)-mediated upregulation of VEGF transcription. In this study, we have tried to decipher the mechanism of E2-promoting angiogenesis. We pre-incubated human endometrial microvascular endothelial cells (HEMECs) with E2 and performed western blotting, qRT-PCR, and cellular immunofluorescence experiments. We observed that E2 treatment of HEMECs increased ERα expression and reduced the expression of GRP78, which led to reduction of Caspase 3 expression by the CHOP pathway. In addition, E2 not only activated ERK signaling pathway but also inhibited p65 phosphorylation along with its translocation from nucleus to the cytoplasm, and subsequently inhibiting GRP78 expression, which led to inhibition of cell apoptosis. Together, these findings highlight the novel mechanism underlying E2-mediated improvement in endometrial angiogenesis through the ERK-p65 signaling pathway.

Selenium ameliorates aflatoxin B1-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells.

This study aimed to observe the effects of Selenium (Se) on aflatoxin B1 (AFB1)-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells (HEMECs). Fifty female mice were randomly divided into control group; AFB1 group; Se group (0.2 mg/kg each day); AFB1 + Se group; and positive control group. After continuous treatment for 30 days, uterine tissues were harvested, which were used for hematoxylin and eosin (H&E) staining. Necrosis-related proteins including RIPK1, RIPK3, and MLKL were examined in uterine tissues using western blot and immunohistochemistry. HEMECs were treated with different concentrations of AFB1 or Se, which were used for selecting the optimal concentrations. RIPK1, RIPK3 and MLKL expression was detected in HEMECs exposed to AFB1 and/or Se via western blot and immunofluorescence. H&E staining results showed that AFB1 induced uterine injury of female male, which was ameliorated by Se treatment. According to western blot and immunohistochemistry, RIPK1, RIPK3, and MLKL expression was distinctly increased in uterine tissues of AFB1-treated mice, which was decreased by Se treatment. Cell viability of HEMECs was gradually lowered as the concentrations of AFB1and Se increased. A 10-μM AFB1 exposure significantly increased RIPK1, RIPK3, and MLKL expression in HEMECs, which was improved following co-treatment with 5-μM Se. Thus, our findings revealed that Se may ameliorate AFB1-induced uterine injury in female mice and necrosis of HEMECs.

Exposure to mono (2-ethylhexyl) phthalate facilitates apoptosis and pyroptosis of human endometrial microvascular endothelial cells through NLRP3 inflammasome.

Mono (2-ethylhexyl) phthalate (MEHP) is a major metabolite of di (2-ethylhexyl) phthalate (DEHP). This study aimed to observe the toxic effect of MEHP on human endometrial microvascular endothelial cells (HEMECs) and its potential molecular mechanism. HEMECs were exposed to different concentrations of MEHP (0, 50, 100, and 200 nM). Cell viability and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry assays. Western blot was performed to examine the expression of apoptosis-related proteins (Bcl-2, Bax, and Caspase-3). Moreover, the expression of pyroptosis-related Caspase-1 was detected by western blot and immunofluorescence assays. Lactate dehydrogenase (LDH) release levels were evaluated in HEMECs treated with MEHP and/or Caspase-1 inhibitor Ac-YVAD-CHO. After exposure to MEHP, NLRP3 expression was examined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. LDH release and apoptosis levels were tested in HEMECs induced by MEHP and/or siNLRP3. MEHP significantly induced cell viability and inhibited apoptosis for HEMECs, with a concentration-dependent manner. Furthermore, Bcl-2/Bax ratio was distinctly reduced and Caspase-3 expression was increased in HEMECs after exposure to MEHP. Western blot and immunofluorescence results confirmed that MEHP markedly augmented Caspase-1 expression in HEMECs. Furthermore, LDH release levels were fortified in HEMECs treated with MEHP, which were improved following cotreatment with Ac-YVAD-CHO. At the mRNA and protein levels, NLRP3 expression was prominently increased in HEMECs exposed to MEHP. NLRP3 knockdown markedly ameliorated the increase in LDH release and apoptosis induced by MEHP exposure in HEMECs. Our findings suggested that exposure to MEHP facilitates apoptosis and pyroptosis of HEMECs through NLRP3 inflammasome.

Bu Shen Zhu Yun Decoction Improves Endometrial Receptivity via VEGFR-2-Mediated Angiogenesis.

Vascular endothelial growth factor receptor-2 (VEGFR-2) regulates the mitogen-activated protein kinase (MAPK) signaling pathway and plays an important role in angiogenesis. Bu Shen Zhu Yun decoction (BSZYD) can improve endometrial receptivity and embryo implantation rates in patients undergoing in vitro fertilization. However, whether BSZYD improves endometrial receptivity via angiogenesis remains unclear. Here, we investigated the effects of BSZYD on the proliferation, migration, and angiogenesis of human endometrial microvascular endothelial cells (HEMECs) and found that BSZYD upregulated the expression of cyclin D1, matrix metalloproteinase 9 (MMP9), and proliferating cell nuclear antigen (PCNA) in HEMECs. Cell Counting Kit 8 assay, scratch-wound assay, and Tube Formation Assay results showed that BSZYD promoted the proliferation, migration, and angiogenesis of HEMECs. Western blot analysis results revealed the activation of the MAPK signaling pathway by BSZYD through the upregulation of VEGF and VEGFR-2 expression. Together, these findings highlight the novel mechanism underlying BSZYD-mediated improvement in endometrial receptivity through the MAPK signaling pathway.

β2-Adrenergic receptor expression in human endometrial endothelial cells potentially mediates 2-hydroxyestradiol-induced microvascular stabilization: implications for endometriosis

Catecholestrogens are biologically active metabolites of 17β-estradiol (E2) synthesized by cytochrome P450 enzymes forming 2-Hydroxyestradiol (OHE2) and 4-OHE2. Although catecholestrogens bind estrogen receptors (ERs) with low affinity, their high affinity for β2-adrenergic receptors (β2-AR) regulates uterine endothelial cell proliferation in gestation1. Women with endometriosis have aberrant E2 metabolizing enzymes potentially inducing catecholestrogen production2. We hypothesize that ectopic and eutopic endometrial microvascular endothelial cells express β2-AR and that, under stimulation by 2-OHE2, endometrial endothelial cells have increased cell viability and an effect on endothelial tube stabilization. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained for β2-AR and evaluated semi-quantitatively by HSCORE. Confluent human endometrial endothelial cells (HEEC) grown in 96-well plates were pretreated with control or with β-AR antagonist propranolol (2x10-6 M) followed by treatment with vehicle (control) or with 10-8 M E2 or 2-OHE2. Then, vascular tube formation (15x103 cells/well, n=3) and endothelial proliferation (5x103 cells/well, n=3) were evaluated. Tube formation was analyzed at 2, 4, 8, 16, and 24h post-treatment. Images were utilized to score endothelial tube formation and degradation based on the following scores: initial tube coalescence= 1, initiation of tube networks= 2, completed tube networks= 3, vascular destabilization= 4, and complete loss of vascular network= 5. XTT assays measured cell viability as an indicator of proliferation at 24h post-treatment. Statistical analysis used a t-test or One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed increased β2-AR staining in vascular endothelial cells in ectopic compared to eutopic tissue during the proliferative phase (P<0.05) and showed a trend toward increased β2-AR staining in eutopic compared to ectopic tissue in the secretory phase; however, it did not achieve significance (P=0.055). In vitro endothelial tube formation analyses revealed no difference in scores at 2, 4, and 8h post-treatment with both E2 and 2-OHE2 compared with control. However, a difference at 16 and 24h post-treatment existed in that treatment with E2 and 2-OHE2 led to lower scores (mean = 3) compared to control (mean = 5) indicating a stabilization of endothelial tube structure in the treatment groups. XTT analyses revealed that compared to control, there were no significant differences in proliferative index in the treatment groups compared to control (P>0.05).Figure 2Endothelial Tube Formation Assay showing control compared to E2 10-8 M or 2-OHE2 10-8 M at hours 2, 4, 8, 16, and 24h post-treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increased β2-AR expression in endothelial cells in ectopic endometriotic tissue during the proliferative phase suggests it plays a role in angiogenesis during implantation of ectopic endometrial tissue. Our in vitro data, that stabilization of newly formed endothelial tube network by 2-OHE2, suggest that the effect of 2-OHE2 may be mediated by β2-AR on endometriotic endothelial cells to maturate newly formed vasculatures during angiogenesis, thereby contributing to endometriosis pathogenesis.

Low molecular weight heparin and aspirin exacerbate human endometrial endothelial cell responses to antiphospholipid antibodies.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications despite treatment with low molecular weight heparin (LMWH) or aspirin (ASA). aPL recognizing beta2 glycoprotein I can target the uterine endothelium, however, little is known about its response to aPL. This study characterized the effect of aPL on human endometrial endothelial cells (HEECs), and the influence of LMWH and ASA. HEECs were exposed to aPL or control IgG, with or without low-dose LMWH and ASA, alone or in combination. Chemokine and angiogenic factor secretion were measured by ELISA. A tube formation assay was used to measure angiogenesis. aPL increased HEEC secretion of pro-angiogenic VEGF and PlGF; increased anti-angiogenic sFlt-1; inhibited basal secretion of the chemokines MCP-1, G-CSF, and GRO-α; and impaired angiogenesis. LMWH and ASA, alone and in combination, exacerbated the aPL-induced changes in the HEEC angiogenic factor and chemokine profile. There was no reversal of the aPL inhibition of HEEC angiogenesis by either single or combination therapy. By aPL inhibiting HEEC chemokine secretion and promoting sFlt-1 release, the uterine endothelium may contribute to impaired placentation and vascular transformation. LMWH and ASA may further contribute to endothelium dysfunction in women with obstetric APS.

The extracellular signal-regulated kinase 1/2 triggers angiogenesis in human ectopic endometrial implants by inducing angioblast differentiation and proliferation.

The role of extracellular signal-regulated kinase (ERK)1/2-mediated angiogenesis during endometriotic nidation is unknown. We posit that ERK1/2-induced angioblast differentiation and proliferation promotes ectopic endometrial angiogenesis. Human eutopic and ectopic endometria were immunostained for total- (T-) or phosphorylated- (P-) ERK1/2 or double-immunostained for P-ERK1/2-CD34 and PCNA-CD34. Estradiol (E2 ), cytokines, normal peritoneal fluid (NPF) or endometriotic peritoneal fluid (EPF) ±PD98059, an ERK1/2 inhibitor, treaded primary human endometrial endothelial cells (HEECs) were evaluated by T-/P-ERK1/2 immunoblotting, MTT viability and tube formation assays. HEECs exhibited higher endothelial P-ERK1/2 immunoreactivity in ectopic vs eutopic endometria. Double-immunostained ectopic endometria displayed abundant CD34-positive angioblasts exhibiting strong P-ERK1/2 and PCNA immunoreactivity. EPF and vascular growth factor (VEGF)-A significantly increased HEEC proliferation and P-ERK1/2 levels. PD98059 reduced basal, EPF, and VEGF-induced HEEC proliferation and promoted vascular stabilization following tube formation. Enhanced ERK1/2 activity in angioblasts by such peritoneal factors as VEGF, E2 induces proliferation to trigger ectopic endometrial angiogenesis.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding

ObjectiveProgestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte–endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. Study designLiquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. ResultsThrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. ConclusionThese results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. ImplicationsMass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

Context:Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined.Objective:To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses.Patients/Setting:Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood.Design:CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture.Results:CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone.Conclusions:These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-μm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.

The Effect of Levonorgestrel on Fibrinolytic Factors in Human Endometrial Endothelial Cells.

The levonorgestrel-releasing intrauterine system is considered a highly effective treatment of heavy menstrual bleeding (HMB). While LNG has established effects on the stromal and glandular compartments of the endometrial tissue, its effect on the endometrial endothelial cells has not been investigated. We examined whether LNG regulates fibrinolytic factors, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) secreted by human endometrial endothelial cells (HEECs) and determined the steroid receptor through which LNG exerts its effect on the endothelium. The HEECs were treated with LNG or progesterone and levels of tPA and plasminogen activator inhibitor 1 (PAI-1) measured. The HEECs were specifically examined for the presence of androgen receptors through Western blot. Levonorgestrel ± flutamide were added to HEECs and the levels of tPA and uPA were examined. An enzyme-linked immunosorbent assay performed on culture media confirmed a statistically significant decrease in tPA levels in cells treated with LNG (77.80% ± 8.0% of control; n = 5, P < .05 vs control) but not progesterone. The androgen receptor (110 kDa) was detected in HEEC lysates. The decrease in tPA was blocked by the addition of flutamide (101.3% ± 16% of control), a classic nonsteroidal androgen receptor blocker. There was no change in uPA or PAI-1 levels in cells treated with LNG. Levonorgestrel decreases tPA levels through the androgen receptor in HEECs. Thus, LNG inhibits tPA secretion by the endometrial endothelial cell. This response suggests reduction in HMB with LNG-IUS could reflect an LNG-mediated promotion of hemostasis.

Thrombin Augments LPS-Induced Human Endometrial Endothelial Cell Inflammation via PAR1 Activation.

Risk factors for preterm birth include placental abruption, giving rise to excessive decidual thrombin, and intrauterine bacterial infection. Human endometrial endothelial cells (HEECs) express Toll-like receptors (TLRs), and infection-derived agonists trigger HEECs to generate specific inflammatory responses. As thrombin, in addition to inducing coagulation, can contribute to inflammation, its effect on HEEC inflammatory responses to the TLR4 agonist, bacterial lipopolysaccharide (LPS), was investigated. HEECs were pre-treated with or without thrombin or specific protease-activated receptor (PAR) agonists, followed by treatment with or without LPS. Supernatants were measured for cytokines and chemokines by ELISA and multiplex analysis. Thrombin significantly and synergistically augmented LPS-induced HEEC secretion of interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), and growth-regulated oncogene-alpha (GRO-α), and significantly augmented monocyte chemotactic protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) secretion additively. Similar to thrombin, a PAR1 agonist synergistically augmented the LPS-induced HEEC secretion of inflammatory IL-6, IL-8, G-CSF, and GRO-α. Thrombin, via PAR1 activation, synergistically augments LPS-induced HEEC production of chemokines involved in immune cell recruitment and survival, suggesting a mechanism by which intrauterine abruption and bacterial infection may together be associated with an aggravated uterine inflammatory response.