Hyaluronic acid-mediated epithelial-mesenchymal transition of human lens epithelial cells via CD44 and TGF-β2

ABSTRACT Purpose: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-β2 in epithelial-mesenchymal transition. However, the role of TGF-β2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-β2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. Methods: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-β2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-β2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. Results: Treatment with hyaluronic acid (1.0 μM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-β2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-β2-mediated epithelial-mesenchymal transition response of HLEB3 cells. Conclusions: Our study showed that both CD44 and TGF-β2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-β2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-β2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.

Unlocking the Secrets of Adipose Tissue: How an Obesity-Associated Secretome Promotes Osteoblast Dedifferentiation via TGF-ß1 Signaling, Paving the Path to an Adipogenic Phenotype.

BACKGROUND: Obesity poses a significant global health challenge, given its association with the excessive accumulation of adipose tissue (AT) and various systemic disruptions. Within the adipose microenvironment, expansion and enrichment with immune cells trigger the release of inflammatory mediators and growth factors, which can disrupt tissues, including bones. While obesity's contribution to bone loss is well established, the direct impact of obese AT on osteoblast maturation remains uncertain. This study aimed to explore the influence of the secretomes from obese and lean AT on osteoblast differentiation and activity. METHODS: SAOS-2 cells were exposed to the secretomes obtained by culturing human subcutaneous AT from individuals with obesity (OATS) or lean patients, and their effects on osteoblasts were evaluated. RESULTS: In the presence of the OATS, mature osteoblasts underwent dedifferentiation, showing an increased proliferation accompanied by a morphological shift towards a mesenchymal phenotype, with detrimental effects on osteogenic markers and the calcification capacity. Concurrently, the OATS promoted the expression of mesenchymal and adipogenic markers, inducing the formation of cytoplasmic lipid droplets in SAOS-2 cells exposed to an adipogenic differentiation medium. Additionally, TGF-ß1 emerged as a key mediator of these effects, as the OATS was enriched with this growth factor. CONCLUSIONS: Our findings demonstrate that obese subcutaneous AT promotes the dedifferentiation of osteoblasts and increases the adipogenic profile in these cells.

Analysis of miR-483-3p and miR-630 expression profile in pediatric adrenocortical tumors and the effect of their modulation on adrenal tumorigenesis in vitro.

Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and outcomes of several types of cancer. However, the role played by miRNAs in pediatric ACT has been poorly explored. In this study, we have evaluated the expression of miR-483-3p and miR-630 in 67 pediatric ACT and 19 non-neoplastic adrenal samples, the effects of the modulations of these miRNAs, and their relationship with the TGF-ß pathway in the H295R and H295A cell lines. Deregulation of both miRNAs was related to survival and disease advanced stages and hence to patients' prognosis. Moreover, modified miR-483-3p and miR-630 in vitro expression decreased cell viability and colony formation capacity, changed how some genes of the TGF-ß pathway, such as TGFBR1, TGFBR2, and SMAD7, are expressed, and altered Smad3, pSmad3, Smad 2/3, N-cadherin, and Vimentin protein expression. Besides that, when inhibition of the TGF-ß pathway was combined with miR-630 overexpression or miR-483-3p silencing, cell viability and colony formation capacity decreased, and protein expression in the TGF-ß pathway changed. Together, the data indicate that both miRNAs participate in the TGF-ß pathway and are therefore potential markers for predicting the prognosis of patients with pediatric ACT.

Neu1 deficiency and fibrotic lymph node microenvironment lead to imbalance in M1/M2 macrophage polarization.

Macrophages play a pivotal role in tissue homeostasis, pathogen defense, and inflammation resolution. M1 and M2 macrophage phenotypes represent two faces in a spectrum of responses to microenvironmental changes, crucial in both physiological and pathological conditions. Neuraminidase 1 (Neu1), a lysosomal and cell surface sialidase responsible for removing terminal sialic acid residues from glycoconjugates, modulates several macrophage functions, including phagocytosis and Toll-like receptor (TLR) signaling. Current evidence suggests that Neu1 expression influences M1/M2 macrophage phenotype alterations in the context of cardiovascular diseases, indicating a potential role for Neu1 in macrophage polarization. For this reason, we investigated the impact of Neu1 deficiency on macrophage polarization in vitro and in vivo. Using bone marrow-derived macrophages (BMDMs) and peritoneal macrophages from Neu1 knockout (Neu1-/- ) mice and wild-type (WT) littermate controls, we demonstrated that Neu1-deficient macrophages exhibit an aberrant M2-like phenotype, characterized by elevated macrophage mannose receptor 1 (MMR/CD206) expression and reduced responsiveness to M1 stimuli. This M2-like phenotype was also observed in vivo in peritoneal and splenic macrophages. However, lymph node (LN) macrophages from Neu1-/- mice exhibited phenotypic alterations with reduced CD206 expression. Further analysis revealed that peripheral LNs from Neu1-/- mice were highly fibrotic, with overexpression of transforming growth factor-beta 1 (TGF-ß1) and hyperactivated TGF-ß signaling in LN macrophages. Consistently, TGF-ß1 was found to alter M1/M2 macrophage polarization in vitro. Our findings showed that Neu1 deficiency prompts macrophages towards an M2 phenotype and that microenvironmental changes, particularly increased TGF-ß1 in fibrotic tissues such as peripheral LNs in Neu1-/- mice, further influence M1/M2 macrophage polarization, highlighting its sensitivity to the local microenvironment. Therapeutic interventions targeting Neu1 or TGF-ß signaling pathways may offer the potential to regulate macrophage behavior across different diseases.

Impact of Plasminogen Activator Inhibitor-1 Serum Levels and the -675 4G/5G Variant in the SERPINE1 Gene on Systemic Sclerosis in a Mexican Population.

Systemic sclerosis (SSc) is characterized by a complex interplay of vascular damage, inflammation, and fibrosis, affecting the skin and internal organs. Plasminogen activator inhibitor-1 (PAI-1), a protein encoded by the SERPINE1 gene, is a potential biomarker of SSc because it is primarily involved in fibrinolysis and is associated with the severity of some autoimmune diseases. This study aimed to determine the association between SERPINE1 variant -675 4G/5G and soluble PAI-1 (sPAI-1) levels with the clinical characteristics and risk of SSc in a Mexican population. This cross-sectional study included 56 SSc patients and 114 control subjects (CSs). The variant was genotyped via the PCR-RFLP method and the levels of sPAI-1 were determined using enzyme-linked immunosorbent assays (ELISAs). The -675 4G/5G variant was not associated with SSc risk or sPAI-I levels. However, higher sPAI-1 levels were observed in SSc patients than in CSs (p = 0.045); these levels were significantly correlated with age, platelets, glucose, and serum levels of transforming growth factor (TGF)-ß1, 2, and 3. The SERPINE1 -675 4G/5G variant did not show any association with SSc risk or sPAI-I levels. However, our study shows a possible alteration of sPAI-1 in this disease, which could be associated with the fibrotic and thrombotic processes in SSc.

Evidence for TGF-ß1/Nrf2 Signaling Crosstalk in a Cuprizone Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-ß1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-ß1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-ß1 receptor reduced Nrf2, catalase, and TGFß-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-ß1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.

OxInflammation Affects Transdifferentiation to Myofibroblasts, Prolonging Wound Healing in Diabetes: A Systematic Review.

Skin wounds, primarily in association with type I diabetes mellitus, are a public health problem generating significant health impacts. Therefore, identifying the main pathways/mechanisms involved in differentiating fibroblasts into myofibroblasts is fundamental to guide research into effective treatments. Adopting the PRISMA guidelines, this study aimed to verify the main pathways/mechanisms using diabetic murine models and analyze the advances and limitations of this area. The Medline (PubMed), Scopus, and Web of Science platforms were used for the search. The studies included were limited to those that used diabetic murine models with excisional wounds. Bias analysis and methodological quality assessments were undertaken using the SYRCLE bias risk tool. Eighteen studies were selected. The systematic review results confirm that diabetes impairs the transformation of fibroblasts into myofibroblasts by affecting the expression of several growth factors, most notably transforming growth factor beta (TGF-beta) and NLRP3. Diabetes also compromises pathways such as the SMAD, c-Jun N-terminal kinase, protein kinase C, and nuclear factor kappa beta activating caspase pathways, leading to cell death. Furthermore, diabetes renders the wound environment highly pro-oxidant and inflammatory, which is known as OxInflammation. As a consequence of this OxInflammation, delays in the collagenization process occur. The protocol details for this systematic review were registered with PROSPERO: CRD42021267776.

The role of osteopontin and osteocyte-derived factors in secondary hyperparathyroidism-induced myopathy.

Muscle weakness is a common symptom in CKD patients, and the pathway by which secondary hyperparathyroidism (SHPT) affects muscle function is unknown. Osteopontin (OPN), a bone matrix protein stimulated by PTH and phosphate, has been associated with inflammatory muscle diseases. In this observational and prospective cohort study, we evaluated 30 patients with severe SHPT (39 ± 12 yr; 18 women), before and 6 mo after parathyroidectomy (PTx). We examined the relationships among CKD-mineral and bone disorder parameters; myokine and inflammatory cytokine levels; and changes in resting energy expenditure (REE), muscle function, BMD, and muscle-related proteins. At baseline, the patients showed low gene expression of muscle turnover markers and irisin, as well as high protein expression of OPN, transforming growth factor beta (TGF-ß), and fibroblast growth factor 21. Six months after PTx, REE and muscle mass had not changed, but physical performance, muscle strength, and bone mass improved, more so in patients undergoing total PTx. Also, there were reductions in the protein expression of OPN (11 vs 3%, p=.01) and TGF-ß (21 vs 7%, p=.002) in muscle, together with a significant increase in irisin muscular levels (30 vs 35 pg/mg, p=.02). The gain in bone mass and the increase in irisin levels correlated with a reduction in PTH. The levels of interleukin (IL)-1ß, tumor necrosis factor alpha, and IL-17 (markers of myositis) were also lower after PTx. Our data suggest that SHPT plays a role in CKD-induced muscle dysfunction, indirectly, via release of bone-specific proteins, which is partially reverted with PTx.

Full-spectrum cannabidiol reduces UVB damage through the inhibition of TGF-ß1 and the NLRP3 inflammasome.

The thermodynamic characteristics, antioxidant potential, and photoprotective benefits of full-spectrum cannabidiol (FS-CBD) against UVB-induced cellular death were examined in this study. In silico analysis of CBD showed antioxidant capacity via proton donation and UV absorption at 209.09, 254.73, and 276.95 nm, according to the HAT and SPLET methodologies. FS-CBD protected against UVB-induced bacterial death for 30 min. FS-CBD protected against UVB-induced cell death by 42% (1.5 µg/mL) and 35% (3.5 µg/mL) in an in vitro keratinocyte cell model. An in vivo acute irradiated CD-1et/et mouse model (UVB-irradiated for 5 min) presented very low photoprotection when FS-CBD was applied cutaneously, as determined by histological analyses. In vivo skin samples showed that FS-CBD regulated inflammatory responses by inhibiting the inflammatory markers TGF-ß1 and NLRP3. The docking analysis showed that the CBD molecule had a high affinity for TGF-ß1 and NLRP3, indicating that protection against inflammation might be mediated by blocking these proinflammatory molecules. This result was corroborated by the docking interactions between CBD and TGF-ß1 and NLRP3, which resulted in a high affinity and inhibition of both proteins The present work suggested a FS-CBD moderate photoprotective agent against UVB light-induced skin damage and that this effect is partially mediated by its anti-inflammatory activity.

Imbalanced IL10/TGF-ß production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis.

BACKGROUND: Human T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-ß. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines. METHODS: Asymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-ß-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells. RESULTS: Total frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group. CONCLUSIONS: A higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-ß may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells.

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-ß or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-ß and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-ß-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-ß. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway.

BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-ß pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-ß pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-ß signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Adenosine Increases the Immunosuppressive Capacity of Cervical Cancer Cells by Increasing PD-L1 Expression and TGF-ß Production through Its Interaction with A2AR/A2BR.

The present study provides evidence showing that adenosine (Ado) increases the expression of programmed death ligand 1 (PD-L1) in cervical cancer (CeCa) cells by interacting with A2AR/A2BR and that TGF-ß1 acts in an autocrine manner to induce PD-L1 expression, enhancing the immunosuppressive effects of CeCa cells on activated T lymphocytes (ATLs) and CD8+ cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from E6 and E7 proteins of HPV-16. Interestingly, the addition of the antagonists ZM241385 and MRS1754, which are specific for A2AR and A2BR, respectively, or SB-505124, which is a selective TGF-ß1 receptor inhibitor, to CeCa cell cultures significantly inhibited PD-L1 expression. In addition, supernatants from CeCa cells that were treated with Ado (CeCa-Ado Sup) increased the expression of PD-1, TGF-ß1, and IL-10 and decreased the expression of IFN-γ in ATLs. Interestingly, the addition of an anti-TGF-ß neutralizing antibody strongly reversed the effect of CeCa-Ado Sup on PD-1 expression in ATLs. These results strongly suggest the presence of a feedback mechanism that involves the adenosinergic pathway, the production of TGF-ß1, and the upregulation of PD-L1 expression in CeCa cells that suppresses the antitumor response of CTLs. The findings of this study suggest that this pathway may be clinically important and may be a therapeutic target.

Doxorubicin-induced chemoresistance in Duke's type B colon adenocarcinoma cell line is aggravated in the presence of TGF-ß2 through non-apoptotic cell death.

BACKGROUND: The current challenge in clinical cancer treatment is chemoresistance. Colon cells have inherently higher xenobiotic transporters expression and hence can attain resistance rapidly. Increased levels of TGF-ß2 expression in patients have been attributed to cancer progression, aggressiveness, and resistance. To investigate resistance progression, we treated doxorubicin (dox) to HT-29 colon adenocarcinoma cells in the presence or absence of TGF-ß2 ligand. METHODS: After 1, 3-, and 7-day treatment, we investigated cell proliferation, viability, and cytotoxicity by MTT, trypan blue staining, and lactate dehydrogenase enzyme release. The mechanism of cell death was elucidated by hoechst33342 and propidium iodide dual staining and apoptosis assay. The development of resistance was detected by rhodamine123 efflux and P-glycoprotein (P-gp)/MDR1 antibody staining through fluorimetry and flow cytometry. The colony formation ability of the cells was also elucidated. RESULTS: Inhibition of cell proliferation was noted after day 1, while a significant reduction in viability and a significant increase in lactate dehydrogenase release was detected after day 3. Reduction of intracellular rhodamine123 levels was detected after day 3 and was significantly lower in dox with TGF-ß2 treatment compared to dox alone. Increased surface P-gp levels after days 3 and 7 were observed in the treated groups. Hoechst33342/propidium iodide staining and apoptosis assay indicated non-apoptotic cell death. The cells treated with TGF-ß2 had higher colony formation ability. CONCLUSIONS: TGF-ß2 expression might play a significant role in the development of chemoresistance to doxorubicin in Duke's type B colon adenocarcinoma cell line, HT-29.

Transforming growth factors ß and their signaling pathway in renal cell carcinoma and peritumoral space-transcriptome analysis.

PURPOSE: The aim of the study was to verify hypotheses: Are transforming growth factors TGFß1-3, their receptors TGFßI-III, and intracellular messenger proteins Smad1-7 involved in the pathogenesis of kidney cancer? What is the expression of genes of the TGFß/Smads pathway in renal cell carcinoma (RCC) tissues, peritumoral tissues (TME; tumor microenvironment), and in normal kidney (NK) tissue?. METHODS: Twenty patients with RCC who underwent total nephrectomy were included into the molecular analysis. The mRNA expression of the genes was quantified by RT-qPCR. RESULTS: The study showed that the expression of the genes of TGFß/Smads pathway is dysregulated in both RCC and the TME: TGFß1, TGFß3 expression is increased in the TME in comparison to the NK tissues; TGFß2, TGFß3, TGFßRI, TGFßRIII, Smad1, Smad2, Smad3, and Smad6 are underexpressed in RCC comparing to the TME tissues; TGFßRI, TGFßRIII, and Smad2 are underexpressed in RCC in comparison to the NK tissues. CONCLUSION: On the one hand, the underexpression of the TGFß signaling pathway genes within the malignant tumor may result in the loss of the antiproliferative and pro-apoptotic activity of this cytokine. On the other hand, the overexpression of the TGFß/Smads pathway genes in the TME than in tumor or NK tissues most probably results in an immunosuppressive effect in the space surrounding the tumor and may have an antiproliferative and pro-apoptotic effect on non-neoplastic cells present in the TME. The functional and morphological consistency of this area may determine the aggressiveness of the tumor and the time in which the neoplastic process will spread.

Effects of calcitriol upon TGF-ßs and their receptors in trophoblast cells.

Calcitriol levels increase during pregnancy, contributing to the hormonal and immunological balance, but its deficiency has been associated with problems during this period. Meanwhile, transforming growth factors-ß (TGF-ßs) play an important role in the maintenance of fetal-maternal immune tolerance; however, exacerbated concentrations of this growth factor are associated with complicated pregnancies. Therefore, we studied the effects of calcitriol on TGF-ßs and their receptors in trophoblast cells. Term placentas from uncomplicated pregnancies after cesarean sections were used for cell cultures. Basal gene expression and the effect of calcitriol upon TGF-ß1, TGF-ß2, TGF-ß3, and their receptors TGF-ßR1 and TGF-ßR2 were assessed using real-time PCR from trophoblast cells. The presence of TGF-ß1, 2, 3, and TGF-ßR1 were evaluated by immunofluorescence, and the protein abundance and secretion of TGF-ß1 were assessed by Western blot and ELISA, respectively. Basal gene expression of TGF-ß1 in trophoblast from term placentas was higher than TGF-ß2 and TGF-ß3, while TGF-ßR2 was higher than TGF-ßR1. The presence and cellular localization of TGF-ß1, 2, 3, and TGF-ßR1 were detected in the cytoplasm of syncytiotrophoblast, with TGF-ß1 showing the highest intensity. Calcitriol significantly inhibited gene expression of TGF-ß1, TGF-ß2, and TGF-ßR1. Likewise, calcitriol decreased the secretion and abundance of TGF-ß1. In conclusion, results indicate that calcitriol is a regulator of TGF-ßs in cultured trophoblast cells from term placentas and therefore may be an important player in the development of healthy pregnancies.

A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-ß and BMP signaling in the Fsh-mediated spermatogonial fate.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-ß superfamily. On the other hand, the specific roles of TGF-ß and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-ß (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-ß or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-ß inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-ß and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-ß and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-ß subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-ß (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

Background Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. Methods Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 μM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 μM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. Results We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. Conclusions Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Kaempferol promotes wound-healing in diabetic rats through antibacterial and antioxidant effects, devoid of proliferative action

The investigation of novel phytochemicals for the prevention and treatment of infections caused by multidrug-resistant pathogens is gaining attention. The current study evaluated the in-vitro antimicrobial activity of kaempferol against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Its in-vivo efficacy in inhibiting these pathogens was determined using an excision wound model in nicotinamide-streptozocin- -induced diabetic rats. Kaempferol displayed an inhibitory effect against the tested bacteria both in vitro and in vivo. It also healed excision wounds at a 1% (w/w) concentration. An increase in antioxidant enzymes in wounded tissue was observed on kaempferol treatment. A reduction in the MRSA and P. aeruginosa counts in wounded tissue together with a reduced epithelization period was observed when compared to the infected control. A thicker epithelium, new capillaries, and a decrease in inflammatory cells were detected by hematoxylin and eosin staining. Furthermore, an increase in collagen fibers and their deposition was observed by Masson's trichrome staining. Kaempferol at 40 µM did not display any toxicity for human keratinocytes grown in media containing high glucose and it did not affect the expression of the pro-healing cytokines genes vascular endothelial growth factor (VEGF) and transforming growth factor-b-1 (TGFß1). Kaempferol displayed antibacterial and antioxidant actions but did not increase the expression of proliferative genes.

TGF-ß/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells.

The TGF-ß and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-ß and Hippo pathways, since TGF-ß modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-ß pathway and TAZ expression and observe that TGF-ß induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-ß/SMAD-signaling, one of the pathways altered in liver cancer.