Selenium elicited an enhanced anti-inflammatory effect in primary bovine endometrial stromal cells with high cortisol background.

BACKGROUND: An elevated endogenous cortisol level due to the peripartum stress is one of the risk factors of postpartum bovine uterine infections. Selenium is a trace element that elicits anti-inflammation and antioxidation properties. This study aimed to reveal the modulatory effect of selenium on the inflammatory response of primary bovine endometrial stromal cells in the presence of high-level cortisol. The cells were subjected to lipopolysaccharide to establish cellular inflammation. The mRNA expression of toll-like receptor 4 (TLR4), proinflammatory factors, and selenoproteins was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blot and immunofluorescence. RESULTS: The pretreatment with sodium selenite (2 and 4 µΜ) resulted in a down-regulation of TLR4 and genes encoding proinflammatory factors, including interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase. Selenium inhibited the activation of NF-κB and the phosphorylation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase. The suppression of those genes and pathways by selenium was more significant in the presence of high cortisol level (30 ng/mL). Meanwhile the gene expression of glutathione peroxidase 1 and 4 was promoted by selenium, and was even higher in the presence of cortisol and selenium. CONCLUSIONS: The anti-inflammatory action of selenium is probably mediated through NF-κB and MAPK, and is augmented by cortisol in primary bovine endometrial stromal cells.

Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis.

BACKGROUND: Patients with endometriosis suffer with chronic pelvic pain and infertility, and from the lack of pharmacologic therapies that consistently halt disease progression. Differences in the endometrium of patients with endometriosis vs. unaffected controls are well-documented. Specifically, shed endometrial tissues (delivered to the pelvic cavity via retrograde menstruation) reveal that a subset of stromal cells exhibiting pro-inflammatory, pro-fibrotic, and pro-senescence-like phenotypes is enhanced in endometriosis patients compared to controls. Additionally, cultured biopsy-derived endometrial stromal cells from endometriosis patients exhibit impaired decidualization, a defined differentiation process required for human embryo implantation and pregnancy. Quercetin, a senolytic agent, shows therapeutic potential for pulmonary fibrosis, a disorder attributed to senescent pulmonary fibroblasts. In rodent models of endometriosis, quercetin shows promise, and quercetin improves decidualization in vitro. However, the exact mechanisms are not completely understood. Therefore, we investigated the effects of quercetin on menstrual effluent-derived endometrial stromal cells from endometriosis patients and unaffected controls to define the signaling pathways underlying quercetin's effects on endometrial stromal cells. METHODS: Menstrual effluent-derived endometrial stromal cells were collected and cultured from unaffected controls and endometriosis patients and then, low passage cells were treated with quercetin (25 µM) under basal or standard decidualization conditions. Decidualization responses were analyzed by measuring the production of IGFBP1 and PRL. Also, the effects of quercetin on intracellular cAMP levels and cellular oxidative stress responses were measured. Phosphokinase arrays, western blotting, and flow cytometry methods were performed to define the effects of quercetin on various signaling pathways and the potential mechanistic roles of quercetin. RESULTS: Quercetin significantly promotes decidualization of control- and endometriosis-endometrial stromal cells. Quercetin substantially reduces the phosphorylation of multiple signaling molecules in the AKT and ERK1/2 pathways, while enhancing the phosphorylation of p53 and total p53 levels. Furthermore, p53 inhibition blocks decidualization while p53 activation promotes decidualization. Finally, we provide evidence that quercetin increases apoptosis of endometrial stromal cells with a senescent-like phenotype. CONCLUSIONS: These data provide insight into the mechanisms of action of quercetin on endometrial stromal cells and warrant future clinical trials to test quercetin and other senolytics for treating endometriosis.

Extracellular vesicles secreted by equine adipose mesenchymal stem cells preconditioned with transforming growth factor ß-1 are enriched in anti-fibrotic miRNAs and inhibit the expression of fibrotic genes in an in vitro system of endometrial stromal cells fibrosis.

Mare endometrosis is a major reproductive problem associated with low fertility and is characterized by persistent inflammation, TGFß-1 signaling, and consequently, extracellular matrix deposition, which compromises endometrial glands. Mesenchymal stem cell-based products (MSCs), such as extracellular vesicles (EVs), have gained attention due to the regulatory effects exerted by their miRNA cargo. Here, we evaluated the impact of preconditioning equine adipose mesenchymal stem cells with TGFß-1 for short or long periods on the anti-fibrotic properties of secreted extracellular vesicles. MSCs were isolated from six healthy horses and exposed to TGFß-1 for 4, 24, and 0 h. The expression of anti-fibrotic and pro-fibrotic miRNAs and mRNAs in treated cells and miRNAs in the cargo of secreted extracellular vesicles was measured. The resulting EVs were added for 48 h to endometrial stromal cells previously induced to a fibrotic status. The expression of anti-fibrotic and pro-fibrotic genes and miRNAs was evaluated in said cells using qPCR and next-generation sequencing. Preconditioning MSCs with TGFß-1 for 4 h enriched the anti-fibrotic miRNAs (mir29c, mir145, and mir200) in cells and EVs. Conversely, preconditioning the cells for 24 h leads to a pro-fibrotic phenotype overexpressing mir192 and mir433. This finding might have implications for developing an EV-based protocol to treat endometrial fibrosis in mares.

IL-10 Enhances the Inhibitory Effect of Adipose-Derived Stromal Cells on Insulin Resistance/Liver Gluconeogenesis by Treg Cell Induction.

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1ß. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1ß, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus.

Red propolis extract associated to platelet-rich plasma and stromal cells with focus in cell therapy and functional tissue regeneration.

The use of platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSC) have been investigated as a form of wound healing enhancement. The objective of this work was to evaluate the association of red propolis (RP) and PRP as inducers of ADSC for application in tissue regeneration. Adipose tissue post-collection and post-cryopreservation was isolated with type II collagenase, characterized by flow cytometry, and differentiated into osteogenic, chondrogenic and adipose cell. The viability of ADSC was evaluated when exposed to different concentrations of RP using the MTT and trypan blue assay. Acridine orange and ethidium bromide (AO/EB) was performed to evaluate cell death events. Horizontal migration methods were investigated in ADSC using autologous and homologous PRP associated with RP (PRP/RP). All assays were processed in triplicate. Flow cytometry and cellular differentiation showed that type II collagenase was effective for isolating ADSC post-collection and post-cryopreservation. RP extracts at concentrations of up to 50 µg.mL-1 presented no cytotoxic effects. Association of PRP and RP at 25 and 50 µg.ml-1 influenced ADSC migration, with total closure on the seventh day after exposition. The results here presented could stimulate proliferation of ADSC cells that may contribute directly or indirectly to the reconstructive process of tissue regeneration.

NK-92 cells activated by IL-2 inhibit the progression of endometriosis in vitro.

BACKGROUND: Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. METHODS: Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. RESULTS: IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. CONCLUSION: IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.

Endometriosis is a common chronic systemic disease affecting approximately 190 million women worldwide. However, clinical treatments for endometriosis remain challenging due to the scarcity of high-quality scientific evidence and conflicting available guidelines. This research was designed to explore whether interleukin (IL)-2 affected the progression of endometriosis by modulating endometrial stromal cell apoptosis and natural killer (NK) cell-mediated cytotoxicity, thereby providing new therapeutic methods for endometriosis.

Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS.

Polycystic ovary syndrome (PCOS) is a complex common endocrine disorder affecting women of reproductive age. Ovulatory dysfunction is recognized as a primary infertile factor, however, even when ovulation is medically induced and restored, PCOS patients continue to experience reduced cumulative pregnancy rates and a higher spontaneous miscarriage rate. Hyperandrogenism, a hallmark feature of PCOS, affects ovarian folliculogenesis, endometrial receptivity, and the establishment and maintenance of pregnancy. Decidualization denotes the transformation that the stromal compart of the endometrium must undergo to accommodate pregnancy, driven by the rising progesterone levels and local cAMP production. However, studies on the impact of hyperandrogenism on decidualization are limited. In this study, we observed that primary endometrial stromal cells from women with PCOS exhibit abnormal responses to progesterone during in vitro decidualization. A high concentration of testosterone inhibits human endometrial stromal cells (HESCs) decidualization. RNA-Seq analysis demonstrated that pyruvate dehydrogenase kinase 4 (PDK4) expression was significantly lower in the endometrium of PCOS patients with hyperandrogenism compared to those without hyperandrogenism. We also characterized that the expression of PDK4 is elevated in the endometrium stroma at the mid-secretory phase. Artificial decidualization could enhance PDK4 expression, while downregulation of PDK4 leads to abnormal decidualization both in vivo and in vitro. Mechanistically, testosterone excess inhibits IGFBP1 and PRL expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. Based on co-immunoprecipitation analysis, we observed an interaction between SIRT1 and PDK4, promoting glycolysis to facilitate decidualization. Restrain of AR activation resumes the AMPK/SIRT1/PDK4 pathway suppressed by testosterone excess, indicating that testosterone primarily acts on decidualization through AR stimulation. Androgen excess in the endometrium inhibits decidualization by disrupting the AMPK/SIRT1/PDK4 signaling pathway. These data demonstrate the critical roles of endometrial PDK4 in regulating decidualization and provide valuable information for understanding the underlying mechanism during decidualization.

Exposure to serum from exclusive heated tobacco product smokers induces mTOR activation and fibrotic features in human cardiac stromal cells.

Chronic smokers have increased risk of fibrosis-related atrial fibrillation. The use of heated-tobacco products (HTPs) is increasing exponentially, and their health impact is still uncertain. We aim to investigate the effects of circulating molecules in exclusive HTP chronic smokers on the fibrotic behavior of human atrial cardiac stromal cells (CSCs). CSCs were isolated from atrial tissue of elective cardiac surgery patients, and exposed to serum lots from young healthy subjects, stratified in exclusive HTP smokers, tobacco combustion cigarette (TCC) smokers, or nonsmokers (NS). CSCs treated with TCC serum displayed impaired migration and increased expression of pro-inflammatory cytokines. Cells cultured with HTP serum showed increased levels of pro-fibrotic markers, and reduced expression of connexin-43. Both TCC and HTP sera increased collagen release and reduced secretion of angiogenic protective factors from CSCs, compared to NS serum. Paracrine support to tube-formation by endothelial cells and to viability of cardiomyocytes was significantly impaired. Treatment with sera of both smokers groups impaired H2O2/NO release balance by CSCs and reduced early phosphorylation of several pathways compared to NS serum, leading to mTOR activation. Cotreatment with rapamycin was able to reduce mTOR phosphorylation and differentiation into aSMA-positive myofibroblasts in CSCs exposed to TCC and HTP sera. In conclusion, the circulating molecules in the serum of chronic exclusive HTP smokers induce fibrotic behavior in CSCs through activation of the mTOR pathway, and reduce their beneficial paracrine effects on endothelial cells and cardiomyocytes. These results point to a potential risk for cardiac fibrosis in chronic HTP users.

Molecular changes in endometrium origin stromal cells during initiation of cardiomyogenic differentiation induced with Decitabine, Angiotensin II and TGF- ß1.

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-ß1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems.

Epidemiologically relevant phthalates affect human endometrial cells in vitro through cell specific gene expression changes related to the cytoskeleton and mitochondria.

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24 h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24 h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed.

[Quercetin Alleviates H2O2-Induced Oxidative Stress Damage to Human Endometrial Stromal Cells by Inhibiting the p38 MAPK/NOX4 Signaling Pathway]. / æ§²çš®ç´ é€šè¿‡æŠ‘åˆ¶p38 MAPK/NOX4信号通路减轻H2O2è¯±å¯¼çš„äººå­å®«å† è†œåŸºè´¨ç»†èƒžæ°§åŒ–åº”æ¿€æŸä¼¤.

Objective: This study aims to systematically evaluate the protective role of quercetin (QCT), a naturally occurring flavonoid, against oxidative damage in human endometrial stromal cells (HESCs) induced by hydrogen peroxide (H2O2). Oxidative stress, such as that induced by H2O2, is known to contribute significantly to cellular damage and has been implicated in various reproductive health issues. The study is focused on investigating how QCT interacts with specific molecular pathways to mitigate this damage. Special attention was given to the p38 MAPK/NOX4 signaling pathway, which is crucial to the regulation of oxidative stress responses in cellular systems. By elucidating these mechanisms, the study seeks to confirm the potential of QCT not only as a protective agent against oxidative stress but also as a therapeutic agent that could be integrated in treatments of conditions characterized by heightened oxidative stress in endometrial cells. Methods: I n vitro cultures of HESCs were treated with QCT at different concentrations (0, 10, 20, and 40 µmol/L) for 24 h to verify the non-toxic effects of QCT on normal endometrial cells. Subsequently, 250 µmol/L H2O2 was used to incubate the cells for 12 h to establish an H2O2-induced HESCs injury model. HESCs were pretreated with QCT for 24 h, which was followed by stimulation with H2O2. Then, CCK-8 assay was performed to examine the cell viability and to screen for the effective intervention concentration. HESCs were divided into 3 groups, the control group, the H2O2 model group, and the H2O2+QCT group. Intracellular levels of reactive oxygen species (ROS) were precisely quantified using the DCFH-DA fluorescence assay, a method known for its accuracy in detecting and quantifying oxidative changes within the cell. The mitochondrial membrane potential was determined by JC-1 staining. Annexin Ⅴ/PI double staining and flow cytometry were performed to determine the effect of QCT on H2O2-induced apoptosis of HESCs. Furthermore, to delve deeper into the cellular mechanisms underlying the observed effects, Western blot analysis was conducted to measure the expression levels of the critical proteins involved in oxidative stress response, including NADPH oxidase 4 (NOX4), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p38 MAPK (p-p38 MAPK). This analysis helps increase understanding of the specific intracellular signaling pathways affected by QCT treatment, giving special attention to its potential for modulation of the p38 MAPK/NOX4 pathway, which plays a significant role in cellular defense mechanisms against oxidative stress. Results: In this study, we started off by assessing the toxicity of QCT on normal endometrial cells. Our findings revealed that QCT at various concentrations (0, 10, 20, and 40 µmol/L) did not exhibit any cytotoxic effects, which laid the foundation for further investigation into its protective roles. In the H2O2-induced HESCs injury model, a significant reduction in cell viability was observed, which was linked to the generation of ROS and the resultant oxidative damage. However, pretreatment with QCT (10 µmol/L and 20 µmol/L) significantly enhanced cell viability after 24 h (P<0.05), with the 20 µmol/L concentration showing the most substantial effect. This suggests that QCT can effectively reverse the cellular damage caused by H2O2. Furthermore, the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H2O2 model group compared to those in the control group (P<0.01). However, co-treatment with QCT significantly reversed this trend (P<0.05), indicating QCT's potential protective role in mitigating cell apoptosis. ROS assays showed that, compared to that in the control group, the average fluorescence intensity of ROS in the H2O2 model group significantly increased (P<0.01). QCT treatment significantly reduced the ROS fluorescence intensity in the H2O2+QCT group compared to the that in the H2O2 model group, suggesting an effective alleviation of oxidative damage (P<0.05). JC-1 staining for mitochondrial membrane potential changes revealed that compared to that in the control, the proportion of cells with decreased mitochondrial membrane potential significantly increased in the H2O2 model group (P<0.01). However, this proportion was significantly reduced in the QCT-treated group compared to that of the H2O2 model group (P<0.05). Finally, Western blot analysis indicated that the expression levels of NOX4 and p-p38 MAPK proteins were elevated in the H2O2 model group compared to those of the control group (P<0.05). Following QCT treatment, these protein levels significantly decreased compared to those of the H2O2 model group (P<0.05). These results suggest that QCT may exert its protective effects against oxidative stress by modulating the p38 MAPK/NOX4 signaling pathway. Conclusion: QCT has demonstrated significant protective effects against H2O2-induced oxidative damage in HESCs. This protection is primarily achieved through the effective reduction of ROS accumulation and the inhibition of critical signaling pathways involved in the oxidative stress response, notably the p38 MAPK/NOX4 pathway. The results of this study reveal that QCT's ability to modulate these pathways plays a key role in alleviating cellular damage associated with oxidative stress conditions. This indicates not only its potential as a protective agent against cellular oxidative stress, but also highlights its potential for therapeutic applications in treating conditions characterized by increased oxidative stress in the endometrium, thereby offering the prospect of enhancing reproductive health. Future studies should explore the long-term effects of QCT and its clinical efficacy in vivo, thereby providing a clear path toward its integration into therapeutic protocols.

An integrative alginate-based 3D in vitro model to explore epithelial-stromal cell dynamics in the breast tumor microenvironment.

The tumor microenvironment (TME) orchestrates cellular and extracellular matrix (ECM) interactions, playing a key role in tumorigenesis, tumor growth, and metastization. Investigating the interplay between stromal-epithelial cells within the TME is paramount for understanding cancer mechanisms but demands reliable biological models. 3D-models have emerged as powerful in vitro tools, but many fall short in replicating cell-cell/cell-matrix interactions. This study introduces a novel hybrid 3D-model of the breast TME, combining epithelial cells, cancer-associated fibroblasts (CAFs), and their ECM. To build the stromal compartment, porous 3D-printed alginate scaffolds were seeded with CAFs, which proliferated and produced ECM. The pores were infused with oxidized peptide-modified alginate hydrogel laden with MCF10A cells, forming the parenchymal compartment. The hybrid system supported epithelial morphogenesis into acini surrounded by fibroblasts and ECM, and could be readily solubilized to recover cells, their matrix, and sequestered soluble factors. Proteome profiling of the retrieved ECM showed upregulation of proteins associated with matrix assembly/remodeling, epithelial-to-mesenchymal transition (EMT), and cancer. The TME-like microenvironment induced a partial EMT in MCF10A cells, generating a hybrid population with epithelial and mesenchymal features, characteristic of aggressive phenotypes. Our model provided new insights into epithelial-stromal interactions within the TME, offering a valuable tool for cancer research in a physiologically-relevant 3D setting.

Breaking Down Barriers in Drug Delivery by Stromal Remodeling Approaches in Pancreatic Cancer.

Pancreatic cancer remains a formidable challenge in oncology due to its aggressive nature and limited treatment options. The dense stroma surrounding pancreatic tumors not only provides structural support but also presents a formidable barrier to effective therapy, hindering drug penetration and immune cell infiltration. This review delves into the intricate interplay between stromal components and cancer cells, highlighting their impact on treatment resistance and prognosis. Strategies for stromal remodeling, including modulation of cancer-associated fibroblasts (CAFs), pancreatic stellate cells (PSCs) activation states, and targeting extracellular matrix (ECM) components, are examined for their potential to enhance drug penetration and improve therapeutic efficacy. Integration of stromal remodeling with conventional therapies, such as chemotherapy and immunotherapy, is discussed along with the emerging field of intelligent nanosystems for targeted drug delivery. This comprehensive overview underscores the importance of stromal remodeling in pancreatic cancer treatment and offers insights into promising avenues for future research and clinical translation.

Effects of carvedilol on human prostate tissue contractility and stromal cell growth pointing to potential clinical implications.

BACKGROUND: Apart from antagonizing ß-adrenoceptors, carvedilol antagonizes vascular α1-adrenoceptors and activates G protein-independent signaling. Even though it is a commonly used antihypertensive and α1-adrenoceptors are essential for the treatment of voiding symptoms in benign prostatic hyperplasia, its actions in the human prostate are still unknown. Here, we examined carvedilol effects on contractions of human prostate tissues, and on stromal cell growth. METHODS: Contractions of prostate tissues from radical prostatectomy were induced by electric field stimulation (EFS) or α1-agonists. Growth-related functions were examined in cultured stromal cells. RESULTS: Concentration-response curves for phenylephrine, methoxamine and noradrenaline were right shifted by carvedilol (0.1-10 µM), around half a magnitude with 100 nM, half to one magnitude with 1 µM, and two magnitudes with 10 µM. Right shifts were reflected by increased EC50 values for agonists, with unchanged Emax values. EFS-induced contractions were reduced by 21-54% with 0.01-1 µM carvedilol, and by 94% by 10 µM. Colony numbers of stromal cells were increased by 500 nM, but reduced by 1-10 µM carvedilol, while all concentrations reduced colony size. Decreases in viability were time-dependent with 0.1-0.3 µM, but complete with 10 µM. Proliferation was slightly increased by 0.1-0.5 µM, but reduced with 1-10 µM. CONCLUSIONS: Carvedilol antagonizes α1-adrenoceptors in the human prostate, starting with concentrations in ranges of known plasma levels. In vitro, effect sizes resemble those of α1-blockers used for the treatment of voiding symptoms, which requires concentrations beyond plasma levels. Bidirectional and dynamic effects on the growth of stromal cells may be attributed to "biased agonism".

Overexpression of USP22 ameliorates LPS-induced endometrial stromal cells inflammation and modulates cells decidualization by inhibiting ferroptosis.

Endometritis and the failure of decidualization of the endometrium are important factors contributing to the increased incidence of abortion. USP22 is associated with various inflammatory diseases and has been shown to be involved in endometrial decidualization in mice. This study aims to investigate whether USP22 is involved in the regulation of inflammatory response and decidualization in human endometrial stromal cells (hESCs). In this study, lipopolysaccharide (LPS) was used to induce inflammation in hESCs, and MPA combined with cAMP was used to induce decidualization of hESCs. USP22 overexpression vector was constructed to study the role of USP22 in endometritis. The results showed that the USP22 protein and mRNA levels were decreased in LPS-induced hESCs. LPS induction increased the levels of TNF-α, IL-1ß, and IL-6, as well as the expression of iNOS and COX2 proteins in hESCs. In the LPS group, the levels of F-actin, PRL, IGFBP1, SLC7A11, and GPX4 proteins decreased, while the levels of lipid peroxidation and total iron content increased. Additionally, the levels of ACSL4 and TFR1 proteins were up-regulated. Overexpression of USP22 reversed LPS-induced cellular inflammation, attenuated decidualization, and inhibited ferroptosis. However, the use of ferroptosis inducers diminished the regulatory effects of USP22 on inflammatory responses and decidualization. In summary, these suggested that USP22 reduces the LPS-induced inflammatory response and regulates the decidualization of hESCs, and possibly involving ferroptosis.

Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and periredoxin 6 in cultured bovine ovarian tissues.

This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200 µg/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800 µg/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800 µg/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400 µg/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800 µg/mL thymol in culture medium increased CAT activity, while 400 or 800 µg/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400 µg/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.

Inhibition and reversal of a TGF-ß1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors.

BACKGROUND: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. METHODS: The transforming growth factor-ß1 (TGF-ß1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. RESULTS: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-ß1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. CONCLUSIONS: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

Comparative cytotoxicity of Acanthamoeba castellanii-derived conditioned medium on human corneal epithelial and stromal cells.

Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.

Zearalenone modulates the function of goat endometrial cells via the mitochondrial quality control system.

Zearalenone (ZEN) is a mycotoxin known for its estrogen-like effects, which can disrupt the normal physiological function of endometrial cells and potentially lead to abortion in female animals. However, the precise mechanism by which ZEN regulates endometrial function remains unclear. In this study, we found that the binding receptor estrogen receptors for ZEN is extensively expressed across various segments of the uterus and within endometrial cells, and a certain concentration of ZEN treatment reduced the proliferation capacity of goat endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Meanwhile, cell cycle analysis revealed that ZEN treatment leaded to cell cycle arrest in goat EECs and ESCs. To explore the underlying mechanism, we investigated the mitochondrial quality control systems and observed that ZEN triggered excessive mitochondrial fission and disturbed the balance of mitochondrial fusion-fission dynamics, impaired mitochondrial biogenesis, increased mitochondrial unfolded protein response and mitophagy in goat EECs and ESCs. Additionally, ZEN treatment reduced the activities of mitochondrial respiratory chain complexes, heightened the production of hydrogen peroxide and reactive oxygen species, and caused cellular oxidative stress and mitochondrial dysfunction. These results suggest that ZEN has adverse effects on goat endometrium cells by disrupting the mitochondrial quality control system and affecting cell cycle and proliferation. Understanding the underlying molecular pathways involved in ZEN-induced mitochondrial dysfunction and its consequences on cell function will provide critical insights into the reproductive toxicity of ZEN and contribute to safeguarding the health and wellbeing of animals and humans exposed to this mycotoxin.

Cabergoline targets multiple pathways to inhibit PRL secretion and increases stromal fibrosis.

OBJECTIVE: Unravel the potential mechanism(s) of the on- and off-target actions of dopamine agonist therapy in both human prolactinoma tumors and neighboring stromal and immune cells. DESIGN AND METHODS: Five surgically resected prolactinomas (PRLomas) from 3 cabergoline (CBG)-treated patients and 2 treatment-naive patients were analyzed by using single-cell RNA sequencing (scRNA-seq) to compare the cellular composition and transcriptional landscape. RESULTS: Six major cell populations, namely tumor (88.2%), immune (5.6%), stromal (4.9%), progenitor cells (0.6%), proliferating cells (0.4%), and erythrocytes (0.2%), were observed. Tumor cells from CBG-treated patients expressed lower levels of genes that regulated hormone secretion, such as SCG2, VGF, TIMP1, NNAT, and CALD1, consistent with the inhibitory effects of CBG on hormone processing and secretion. Interestingly, we also observed an increased number of CD8+ T cells in the CBG-treated tissues. These cytotoxic CD8+ T cells expressed killing granule components such as perforin and the granzymes GZMB, GNLY, and KLRD1 as well as the inflammatory cytokine CCL5. Immune cell activation of these CD8+ T cells was further analyzed in a compartment-specific manner, and increased CD25 (IL2R) expression was noted in the CD8+ T cells from the CBG-treated samples. Additionally, and confirming prior reports, we noted a higher stromal cell population in the CBG-treated samples. CONCLUSIONS: Our scRNA-seq studies revealed key differences in the transcriptomic features of CBG-treated and CBG-untreated PRLomas in both tumor and microenvironment cellular constituents, and for the first time, describe the previously unknown activation of CD8+ T cells following CBG treatment, which may play a role in the tumoricidal actions of CBG.