Adipocyte coculture induces a pro-inflammatory, fibrotic, angiogenic, and proliferative microenvironment in uterine leiomyoma cells.

OBJECTIVE: Obesity and its consequences are among the biggest challenges facing the healthcare system. Uterine leiomyomas are the most common gynecologic tumors. The risk of leiomyoma increases with obesity, but the underlying mechanisms of this association remain unclear. The aim of the present study to determine the cellular and molecular mechanisms by which adipocyte contributes to both leiomyoma tumor initiation and promotion. METHODS: Primary myometrium and leiomyoma cells were isolated from patients who underwent a hysterectomy or myomectomy. Pro-inflammatory, fibrotic, and angiogenic factors were measured using a multiplex cytokine array in human primary and immortalized myometrial and leiomyoma cells cocultured with human adipocyte (SW872) cells, or in animal ELT3 leiomyoma cells cocultured with 3 T3-L1 adipocytes. The free fatty acids (FFAs) and fatty acid-binding protein 4 (FABP4) levels were measured using immunofluorescence assays. Other protein abundances were determined using western blots. The expression levels of TNF-α, MCP-1, phospho-NF-κB, TGFß3 and VEGF-A in lean and obese in different leiomyoma patients were determined by immunofluorescence staining. RESULTS: Adipocytes promote inflammation, fibrosis, and angiogenesis in uterine leiomyoma cells by upregulating associated factors, such as IL-1ß, TNF-α, MCP-1, GM-CSF, TGF-ßs, PLGF, VEGF, HB-EGF, G-CSF and FGF2. Coculture led to the transfer of FFAs and FABP4 from adipocytes to leiomyoma cells, suggesting that adipocytes may modulate metabolic activity in these tumor cells. Increased levels of FFA and FABP4 expressions were detected in obese leiomyoma tissue compared to lean. The adipocyte-leiomyoma cell interaction increased the phospho-NF-κB level, which plays a key role in inflammation, restructuring metabolic pathways, and angiogenesis. Obese leiomyoma patients expressed a higher amount of TNF-α, MCP-1, phospho-NF-κB, TGFß3 and VEGF-A than lean leiomyoma patients, consistent with in vitro findings. Furthermore, we found that adipocyte secretory factors enhance leiomyoma cell proliferation by increasing PCNA abundance. Finally, the inhibition of the inflammatory factors TNF-α, MCP-1, and NF-κB abrogated the adipocyte coculture-induced proliferation of leiomyoma cells. CONCLUSIONS: Adipocytes release inflammatory, fibrotic, and angiogenic factors, along with FFAs, which contribute to a tumor-friendly microenvironment that may promote leiomyoma growth and can represent a new target for leiomyoma prevention and treatment.

Simvastatin reduces plasma membrane caveolae and caveolin-1 in uterine leiomyomas.

AIMS: Uterine leiomyomas, or fibroids, are estrogen dependent benign tumor in women, however, they have limited treatment options. Simvastatin, a drug commonly used to treat high cholesterol. Recently we demonstrated that simvastatin alters estrogen signaling by reducing the expression and trafficking of the estrogen receptor-α (ER-α) in human uterine leiomyoma cells. Caveolae are invaginations of the plasma membrane where ER-α is known to localize and directly interacts with the caveolar protein caveolin-1 (CAV1). This study examines the effects of simvastatin on plasma membrane caveolae and the expression and palmitoylation of CAV1 in human leiomyomas which may influence ER-α signaling. MAIN METHODS: We performed in vitro experiments using primary and immortalized human uterine leiomyoma cells. The caveolae were quantified using transmission electron microscopy. Additionally, we examined the impact of simvastatin treatment (40 mg orally per day for 12 weeks) on human leiomyoma tissue obtained from a randomized controlled trial. The CAV1 protein and mRNA levels were determined using quantitative real-time polymerase chain reactions, western blotting, and immunofluorescence analyses. KEY FINDINGS: Simvastatin decreased the number of caveolae in primary leiomyoma cells and reduced CAV1 abundance in whole cells and remarkably the plasma protein fraction. It also decreased CAV1 palmitoylation, a post-translational modification associated with CAV1 activation. The effects of simvastatin on CAV1 were recapitulated in human leiomyoma tissue samples. SIGNIFICANCE: Our results identify caveolae and CAV1 as novel targets of simvastatin which may contribute to the recently described effects of simvastatin on ER-α signaling and plasma membrane trafficking.

Leptin induces leiomyoma cell proliferation and extracellular matrix deposition via JAK2/STAT3 and MAPK/ERK pathways.

OBJECTIVE: To investigate the molecular effects of leptin on uterine leiomyoma cells. DESIGN: Experimental study using in vitro culture of immortalized human leiomyoma (HuLM) cells. SETTING: Academic university center. PATIENT(S): Women with uterine fibroids who underwent a hysterectomy or myomectomy. INTERVENTION(S): Administration of human recombinant leptin to the media of cultured HuLM cells separately or in combination with pharmacologic Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitors. MAIN OUTCOME MEASURE(S): We examined HuLM tissues and cells for the expression of the leptin receptor, termed OB-R. Cellular proliferation was measured at 6, 24, and 48 hours using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Protein expression levels of proliferating cell nuclear antigen, collagen 1, phosphorylated STAT3/total STAT3, and phosphorylated ERK1/2 and total ERK1/2 were quantified using immunoblotting. Pharmacologic inhibitors were employed to further assess the role of the JAK2/STAT3 and MAPK/ERK pathways in the proliferative response. RESULT(S): The presence of OB-R was confirmed in clinical leiomyoma and myometrial tissue obtained from 3 separate human subjects using immunofluorescence staining, and the expression of OB-R in HuLM cells was identified using immunoblotting. There was no significant difference in the expression of the leptin receptor in the myometrium compared with that in the leiomyoma tissue. Leptin stimulated cell proliferation and extracellular matrix (ECM) deposition at 24 hours after treatment. Pretreatment with a JAK2/STAT3 inhibitor attenuated ECM deposition, and pretreatment with a MAPK/ERK inhibitor significantly decreased leptin's stimulatory effect on cell proliferation and ECM deposition. CONCLUSION(S): Leptin induces a proliferative response and ECM deposition in HuLM cells. These findings suggest that leptin, acting through the JAK2/STAT3 and MAPK/ERK pathways, is involved in the development of uterine leiomyomas, which may partly explain their increased incidence in obese women.

Hypoxia induces proliferation via NOX4-Mediated oxidative stress and TGF-ß3 signaling in uterine leiomyoma cells.

Uterine leiomyomas, the most common tumors of the female reproductive system, are known to have a hypoxic microenvironment. However, the role of such environment in leiomyoma pathobiology remains unknown. The objective was to determine the effects of hypoxia on leiomyoma cells, and the mechanisms. We found that hypoxia induces proliferation and inhibits apoptosis in human leiomyoma cells. This pro-proliferative effect was accompanied by an increase in reactive oxygen species (ROS) generation and the expression of NADPH oxidase 4 (NOX4). The specific NOX4 inhibitor GLX351322 abrogated this hypoxia-induced ROS generation, cellular proliferation, and apoptosis inhibition. To further investigate the mechanism of NOX4-mediated proliferation, we treated leiomyoma cells grown in normoxia with media from leiomyoma cells cultured under hypoxia. This resulted in increased ROS generation and NOX4 expression, suggesting the hypoxia-induced effects are mediated by an autocrine mechanism. We worked to identify the nature of this autocrine factor. We found that the expression of TGF-ß3 and its downstream signaling target pSmad3, are increased in hypoxic leiomyoma cells. To examine the hypothesis that TGF-ß3 is, at least, a part of this autocrine mechanism, we treated hypoxic leiomyoma cells with the HIF-1α inhibitor KC7F2 which we discovered to ameliorate the hypoxia-induced TGF-ß3 expression. Furthermore, pharmacologic inhibition with the TGF-ß/Smad inhibitor SB431542 reduced hypoxia-induced NOX4 expression and ROS generation and attenuated cell proliferation. Thus, we have identified a novel mechanism by which hypoxia induces proliferation in leiomyoma cells. This finding adds to our understanding of leiomyoma pathobiology and can help in identifying new therapeutic targets.

Differential response to hypoxia in leiomyoma and myometrial cells.

AIMS: Recent evidence suggests that repetitive hypoxia occurs during menstrual cycles due to vasoconstriction and myometrial contraction. It is unknown if hypoxia contributes to the development of uterine leiomyoma, the most common tumor of the female reproductive system. This study aims to characterize the response to hypoxia in leiomyoma and myometrial cells; and determine if an aberrant leiomyoma response to hypoxia may contribute to leiomyomatogenesis. MAIN METHODS: Primary and immortalized leiomyoma and myometrial cells were cultured under normoxic and hypoxic conditions. Expression levels of vascular endothelial growth factor-A (VEGF-A), adrenomedullin (ADM), endothelin-1 (ET-1), and hypoxia-inducible factor-1 alpha (HIF-1α) were measured by qRT-PCR, western blotting and ELISA. Cell proliferation was assessed using MTT assay and proliferating-cell-nuclear-antigen (PCNA) expression. KC7F2 (HIF-1α inhibitor) was used to examine the regulating mechanisms. KEY FINDINGS: As expected, hypoxia induced HIF-1α expression in both leiomyoma and myometrial cells. However, hypoxia induced VEGF-A, ET-1 and ADM expression and VEGF-A secretion into the culture media in leiomyoma but not myometrial cells. MTT assay and PCNA expression showed that hypoxia induces proliferation in leiomyoma, but not myometrial cells. HIF-1α inhibitor abrogated the hypoxia-induced VEGF-A, ET-1, ADM, and PCNA expression in leiomyoma cells. SIGNIFICANCE: This study suggests an aberrant leiomyoma cellular response to hypoxia compared to myometrium. This differential response to menstruation-related repetitive hypoxia episodes may lead to selective proliferation of hypoxia-adaptive leiomyoma cells and contribute to leiomyoma growth. Thus, in addition to adding to our understanding of leiomyoma pathobiology, the study proposes angiogenic factors as a potential leiomyoma therapeutic target.

Simvastatin modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradation.

Uterine leiomyomas or fibroids are the most common tumors of the female reproductive tract. Estrogen (E2), a steroid-derived hormone, and its receptors (ERs), particularly ER-α, are important drivers for the development and growth of leiomyomas. We previously demonstrated that simvastatin, a drug used for hyperlipidemia, also possesses anti-leiomyoma properties. The aim of this work is to investigate the impact of simvastatin on ER-α signaling in leiomyoma cells, including its expression, downstream signaling, transcriptional activity, post-translational modification, trafficking and degradation. Primary and immortalized human uterine leiomyoma (HuLM) cells were used for in vitro experiments. Immunodeficient mice xenografted with human leiomyoma tissue explants were used for in vivo studies. Leiomyoma samples were obtained from patients enrolled in an ongoing double-blinded, phase II, randomized controlled trial. Here, we found that simvastatin significantly reduced E2-induced proliferation and PCNA expression. In addition, simvastatin reduced total ER-α expression in leiomyoma cells and altered its subcellular localization by inhibiting its trafficking to the plasma membrane and nucleus. Simvastatin also inhibited E2 downstream signaling, including ERK and AKT pathways, E2/ER transcriptional activity and E2-responsive genes. To explain simvastatin effects on ER-α level and trafficking, we examined its effects on ER-α post-translational processing. We noticed that simvastatin reduced ER-α palmitoylation; a required modification for its stability, trafficking to plasma membrane, and signaling. We also observed an increase in ubiquitin-mediated ER-α degradation. Importantly, we found that the effects of simvastatin on ER-α expression were recapitulated in the xenograft leiomyoma mouse model and human tissues. Thus, our data suggest that simvastatin modulates several E2/ER signaling targets with potential implications in leiomyoma therapy and beyond.

Diet and Nutrition in Gynecological Disorders: A Focus on Clinical Studies.

A healthy lifestyle and a balanced diet play a paramount role in promoting and maintaining homeostatic functions and preventing an array of chronic and debilitating diseases. Based upon observational and epidemiological investigations, it is clear that nutritional factors and dietary habits play a significant role in gynecological disease development, including uterine leiomyoma, endometriosis, polycystic ovary syndrome, and gynecological malignancies. Diets rich in fruits and vegetables, Mediterranean diets, green tea, vitamin D, and plant-derived natural compounds may have a long-term positive impact on gynecological diseases, while fats, red meat, alcohol, and coffee may contribute to their development. Data regarding the association between dietary habits and gynecological disorders are, at times, conflicting, with potential confounding factors, including food pollutants, reduced physical activity, ethnic background, and environmental factors limiting overall conclusions. This review provides a synopsis of the current clinical data and biological basis of the association between available dietary and nutritional data, along with their impact on the biology and pathophysiology of different gynecological disorders, as well as an outlook on future directions that will guide further investigational research.

Vascular biology of uterine fibroids: connecting fibroids and vascular disorders.

Fibroids are benign tumors caused by the proliferation of myometrial smooth muscle cells in the uterus that can lead to symptoms such as abdominal pain, constipation, urinary retention, and infertility. While traditionally thought of as a disease process intrinsic to the uterus, accumulating evidence suggests that fibroid growth may be linked with the systemic vasculature system, although cell-intrinsic factors are certainly of principal importance in their inception. Fibroids are associated with essential hypertension and preeclampsia, as well as atherosclerosis, for reasons that are becoming increasingly elucidated. Factors such as the renin-angiotensin-aldosterone system, estrogen, and endothelial dysfunction all likely play a role in fibroid pathogenesis. In this review, we lay out a framework for reconceptualizing fibroids as a systemic vascular disorder, and discuss how pharmaceutical agents and other interventions targeting the vasculature may aid in the novel treatment of fibroids.

Uterine Stem Cells and Benign Gynecological Disorders: Role in Pathobiology and Therapeutic Implications.

Stem cells in the endometrium and myometrium possess an immense regenerative potential which is necessary to maintain the menstrual cycle and support pregnancy. These cells, as well as bone marrow stem cells, have also been implicated in the development of common benign gynecological disorders including leiomyomas, endometriosis and adenomyosis. Current evidence suggests the conversion of uterine stem cells to tumor initiating stem cells in leiomyomas, endometriosis stem cells, and adenomyosis stem cells, acquiring genetic and epigenetic alterations for the progression of each benign condition. In this comprehensive review, we aim to summarize the progress that has been made to characterize the involvement of stem cells in the pathogenesis of benign gynecologic conditions which, despite their enormous burden, are not yet fully understood. We focus on the stem cell characteristics and aberrations that contribute to the development of benign gynecological disorders and the possible clinical implications of what is known so far. Lastly, we discuss the role of uterine stem cells in the setting of regenerative medicine, particularly in the treatment of Asherman syndrome.Graphical abstract.