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1.
Methods Mol Biol ; 2598: 123-140, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36355289

RESUMO

Extracellular vesicles (EVs) have the capacity for use in cartilage tissue engineering by stimulating tissue repair and microenvironmental reprogramming. This makes them ideal candidates for treating focal cartilage defects and cartilage degeneration in osteoarthritis (OA). Observational studies have reported beneficial biological effects of EVs, such as inhibition of inflammation, enhanced extracellular matrix deposition, and reduced cartilage degradation. Isolation of EVs derived from different source materials such as conditioned cell culture media or biofluids is essential to attribute observed biological effects to EVs as genuine effectors. This chapter presents a density- and a size-based method as well as a combination of both for isolation of EVs from conditioned cell culture media or biofluids. In addition, three methods for characterization of isolated EVs are suggested based on physical properties, protein profiling, and ultrastructural morphology.


Assuntos
Vesículas Extracelulares , Osteoartrite , Humanos , Engenharia Tecidual/métodos , Cartilagem/metabolismo , Osteoartrite/terapia , Osteoartrite/metabolismo , Vesículas Extracelulares/metabolismo , Matriz Extracelular/metabolismo , Meios de Cultivo Condicionados/metabolismo , Condrócitos/metabolismo
2.
Front Endocrinol (Lausanne) ; 13: 989523, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36329893

RESUMO

Objective: Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. Methods: Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. Results: Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. Conclusion: Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive.


Assuntos
Adipócitos , Neoplasias Ovarianas , Humanos , Camundongos , Animais , Feminino , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Adipócitos/metabolismo , Hipóxia/metabolismo , Hipóxia/patologia , Proliferação de Células , Lipídeos/farmacologia , Neoplasias Ovarianas/metabolismo , Microambiente Tumoral
3.
Sci Rep ; 12(1): 18884, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36344527

RESUMO

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1ß, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1ß, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.


Assuntos
NF-kappa B , Fármacos Neuroprotetores , Humanos , Animais , Camundongos , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/metabolismo , Interleucina-6/metabolismo , Proteínas Klotho , Neuroglia/metabolismo , Neurônios/metabolismo , Anti-Inflamatórios/farmacologia , Inflamação/metabolismo
4.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 38(4): 289-294, 2022 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-36414548

RESUMO

Objective: To investigate the protective effects and potential mechanisms of estrogen modified human bone marrow mesenchymal stem cells (hBMSC) on high glucose (HG)-induced injury of vascular endothelial cells. Methods: hBMSCs were cultured under 30 mmol/l glucose to establish a high glucose model (HG), and then were divided into four groups as following: HG group (HG control, without any treatment), HG+E2 group (cells were treated with 20 µmol/L estrogen), HG+E2+ Triciribine group (cells were pretreated with 5 µmol/L protein kinase B (PKB/Akt) inhibitor for 45 min, and then modified by 20 µmol/L estrogen), and NG group (cells were cultured under normal conditions). After 12 h treatment, the cell viability of hBMSC was detected by CCK8 assay, and the contents of NO, VEGF and IL8 in the supernatant of cultured medium in each group were detected by nitrate reductase and ELISA assay (n=6). After 48 h, the expression levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) were detected by Western blot (n=3). In addition, the cell supernatant of each group was further extracted as conditioned medium to culture HUVECs, and the cells were subsequently divided into HG-CM group (HUVECs were treated with HG group's conditioned medium), HG+E2-CM group (HUVECs were treated with HG+E2 group's conditioned medium), HG+E2+Triciribine-CM group (HUVECs were treated with HG+E2+ Triciribine group's conditioned medium) and HG-H group (HUVEC were cultured under HG condition, which were treated with final concentration 30 mmol/l glucose). The cell viability of HUVECs in each group was detected by CCK8 assay after 12 h cultured (n=6). After 24 h treatment, the apoptosis rate of HUVECs in each group was detected by flow cytometry (n=3). Furthermore, the migration rate of HUVECs in each group was observed by wound healing assay after 48 h cultured (n=3). Results: Compared with NG group, the cell viability and eNOS protein phosphorylation level of hBMSC in HG group and the contents of NO, VEGF and IL-8 in the supernatant of cultured medium were decreased (P<0.05). Compared with HG group, the cell viability and eNOS protein phosphorylation level in HG+E2 group and the contents of NO, VEGF and IL-8 in cultured medium supernatant were increased significantly (P<0.05), whereas pre-treatment of hBMSC cells with a Akt inhibitor Triciribine, the above indexes showed reverse changes (P<0.05). Furthermore, compared with HG-CM group, the cell viability and migration ability (P<0.05) of HUVECs in HG+E2-CM group were increased significantly (P<0.05), and the proportion of apoptosis was decreased (P<0.05). While compared with HG+E2-CM group, the cell viability and migration ability of HUVECs in HG+E2+Triciribine-CM group were decreased (P<0.05), and the proportion of apoptosis was increased (P<0.05). Conclusion: Estrogen may promote the secretion of NO, VEGF and IL-8 by activating the Akt/eNOS signaling pathway of hBMSC cells, increase the cell viability and migration ability of HUVECs and inhibit the occurrence of apoptosis, play a protective role against the injury of HUVECs induced by HG condition.


Assuntos
Células-Tronco Mesenquimais , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Endoteliais da Veia Umbilical Humana , Fator A de Crescimento do Endotélio Vascular/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Interleucina-8/metabolismo , Glucose/metabolismo , Estrogênios/farmacologia , Estrogênios/metabolismo
5.
Mol Med ; 28(1): 137, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36401163

RESUMO

BACKGROUND: Intestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism. METHODS: Cecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated. RESULTS: In vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6877002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1ß while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs. CONCLUSIONS: The inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.


Assuntos
Ligante de CD40 , Sepse , Humanos , Camundongos , Animais , Ocludina/metabolismo , Ligante de CD40/metabolismo , Células CACO-2 , S-Nitrosoglutationa/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , RNA Interferente Pequeno , Meios de Cultivo Condicionados/metabolismo , Ativação Plaquetária , Sepse/metabolismo , Neuroglia/metabolismo , Proteínas de Junções Íntimas/metabolismo
6.
Sci Rep ; 12(1): 18718, 2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36333586

RESUMO

Repeat-associated non-AUG translation (RAN translation) is observed in transcripts that are causative for polyglutamine (polyQ) diseases and generates proteins with mono amino acid tracts such as polyalanine (polyA), polyleucine (polyL) and polyserine (polyS) in neurons, astrocytes and microglia. We have previously shown that microglia with aggregated polyQ led to defective differentiation and degeneration of neuron-like cells. However, it has not been determined whether only microglia containing a specific RAN product, but not other RAN products, is harmful in vitro and in vivo. Here we show that polyL-incorporating microglia specifically led to altered startle response in mice. Aggregated polyA, polyS and polyL induced aberrant differentiation of microglia-like BV2 cells. Differentiated PC12 cells treated with conditioned medium (CM) of polyS- and polyL- but not polyA-incorporating microglia-like BV2 cells showed retraction of neurites and loss of branch of neurites. Injection of the polyL-CM, but not polyA-CM and polyS-CM, into the lateral ventricle lowered startle response in mice. Consistently, polyL induced the highest expression of CD68 in BV2 cells. The lowered startle response was replicated in mice given the polyL-CM in the caudal pontine reticular nucleus (PnC), the key region of startle response. Thus, endogenous RAN proteins having polyL derived from polyQ diseases-causative genes in microglia might specifically impair startle response.


Assuntos
Microglia , Reflexo de Sobressalto , Ratos , Camundongos , Animais , Microglia/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Células PC12
7.
PLoS One ; 17(10): e0274056, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36194602

RESUMO

Developing dendritic cells (DCs) from monocytes is a sensitively regulated process. One possible way for cancers to avoid immune recognition and antitumor response is the modulation of DC differentiation. Although several studies are available on the examination of tumor-associated macrophages, a comprehensive analysis focusing on the effects of tumor-formed DCs is not known to date. We provide a comparative analysis of the tumor-edited-monocyte derived DCs differentiated in the presence of adenocarcinomas (MDA, HT29, HeLa)- and primary (WM278, WM983A) or metastatic (WM1617, WM983B) melanomas. The immunomodulatory effect of tumors is mediated at least partly by secreted mediators. We investigated the impact of tumor cell-derived conditioned media on the differentiation of DCs from CD14+ monocytes, sequentially determining the phenotype, cytokine production, phagocytic, and the T cell polarizing capacity of moDCs. We completed our observations by analyzing our data with bioinformatic tools to provide objective correlations between phenotypical and functional properties of different tumor-educated moDCs. The correlation analysis revealed significant differences in the characteristics of adenocarcinomas- or melanomas-edited moDCs. We highlight the functional differences in the properties of moDCs differentiated in the presence of various cancer cell lines. We offer new information and options for the in vitro differentiation protocols of various tumor-conditioned moDCs. Our results confirm that various immunomodulatory properties of different tumor cell lines result in multiple manipulations of DC differentiation.


Assuntos
Adenocarcinoma , Melanoma , Adenocarcinoma/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Células Dendríticas , Humanos , Melanoma/metabolismo , Monócitos/metabolismo
8.
Biol Sex Differ ; 13(1): 56, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36183096

RESUMO

BACKGROUND: Osteocytic microRNA21 (miR21) removal alters cytokine production and bone mass by modulating osteoclast and osteoblast differentiation and activity. Removing osteocytic miR21 increases osteoclast/osteoblast numbers and bone mass in male mice, whereas it decreases osteoclasts/osteoblasts without affecting bone mass in female mice. On the other hand, it leads to sex-independent increases in bone mechanical properties. Because changes in bone remodeling and strength affect skeletal muscle through bone-muscle crosstalk, we investigated whether osteocytic miR21 deletion influences skeletal muscle. METHODS: miR21fl/fl mice and 8kbDMP1-Cre mice were mated to obtain miR21-deficient mice primarily in the osteocyte (OtmiR21Δ) and littermate controls (miR21fl/fl). Four-month-old male and female mice were analyzed. Body composition was examined by DXA/Piximus and gene expression was assessed by qPCR. Ex vivo cultures of long bones devoid of bone-marrow cells from male and female 4-month-old were maintained for 48 h. Conditioned media were collected and used for the C2C12 assays. Two-way ANOVA analyses were performed to determine the contributions of genotype and sex and their interaction to the effects of miR21 deficiency. RESULTS: Lean body mass was increased only in female OtmiR21Δ mice, although miR21 levels in soleus muscle were similar in miR21fl/fl (0.05 ± 0.02) and OtmiR21Δ (0.09 ± 0.04) mice. Female, but not male, OtmiR21Δ mice exhibited increased soleus (42%) and gastrocnemius (21%) muscle weight compared to miR21fl/fl littermates. However, muscle strength and gastrocnemius muscle fiber cross-sectional area were unaltered for either sex. Kinase phosphorylation (phospho/total protein ratio) in soleus muscle, measured as a surrogate for kinase activity by means of multiplex analysis, was also selectively changed depending on the mouse sex. Thus, female OtmiR21Δ mice had higher T185/Y187-ERK1/2 but lower S473-Akt phosphorylation than miR21fl/fl controls, while male OtmiR21Δ mice had higher S473-Akt phosphorylation, suggesting sex-dimorphic shifts in anabolic vs. catabolic signaling. Consistently, levels of FOXO3 and MuRF-1, known to be regulated by Akt, were only increased in male OtmiR21Δ mice. Atrogin-1 mRNA levels were upregulated in female OtmiR21Δ mice, suggesting a potential shift in protein regulation. Sex-specific effects were also found by exposing myotube cultures to conditioned media from 48-h-cultured marrow-flushed bones. Thus 5-day differentiated C2C12 myotubes treated with conditioned media of female OtmiR21Δ mice exhibit 12% higher average diameter compared to cells exposed to miR21fl/fl bone conditioned media. Yet, conditioned media from male bones had no effect on myotube size. CONCLUSIONS: We present a novel aspect of bone-muscle crosstalk in which osteocyte-derived miR21 influences skeletal muscle size, but not strength, in female but not male mice; whereas, intracellular signaling alterations resulting from loss of miR21 seem to alter protein dynamics in a sex-dimorphic fashion.


Assuntos
MicroRNAs , Osteócitos , Animais , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Feminino , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Osteócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo
9.
Nat Commun ; 13(1): 5786, 2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36184639

RESUMO

Spinal cord injury (SCI) triggers neuroinflammation, and subsequently secondary degeneration and oligodendrocyte (OL) death. We report that the alarmin interleukin (IL)-1α is produced by damaged microglia after SCI. Intra-cisterna magna injection of IL-1α in mice rapidly induces neutrophil infiltration and OL death throughout the spinal cord, mimicking the injury cascade seen in SCI sites. These effects are abolished through co-treatment with the IL-1R1 antagonist anakinra, as well as in IL-1R1-knockout mice which demonstrate enhanced locomotor recovery after SCI. Conditional restoration of IL-1R1 expression in astrocytes or endothelial cells (ECs), but not in OLs or microglia, restores IL-1α-induced effects, while astrocyte- or EC-specific Il1r1 deletion reduces OL loss. Conditioned medium derived from IL-1α-stimulated astrocytes results in toxicity for OLs; further, IL-1α-stimulated astrocytes generate reactive oxygen species (ROS), and blocking ROS production in IL-1α-treated or SCI mice prevented OL loss. Thus, after SCI, microglia release IL-1α, inducing astrocyte- and EC-mediated OL degeneration.


Assuntos
Interleucina-1alfa , Traumatismos da Medula Espinal , Alarminas/metabolismo , Animais , Astrócitos/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo
10.
Biochem Biophys Res Commun ; 635: 161-168, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36274366

RESUMO

Mild heat stimulation is defined as a stimulation that occurs several degrees above optimal cell culture or body temperatures. Muscle hypertrophy in C2C12 cells is reportedly facilitated by 39 °C mild heat stimulation. However, the underlying molecular mechanisms by which 39 °C mild heat stimulation promotes muscle hypertrophy remains elusive. In this study, we aimed at understanding the details of these mechanisms. First, we found that 39 °C mild heat stimulation has little or no effect on Myogenesis-Related Factor (MRF) expression both in C2C12 and mouse primary satellite cells. Therefore, we hypothesized that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, essential components in myogenesis. Interestingly, we observed muscle hypertrophy in the group cultured at 37 °C in conditioned medium from 39 °C compared to the group cultured at 37 °C in conditioned medium from 37 °C in the case of both C2C12 and mouse primary satellite cells. These results suggest that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, once released in the culture medium. Finally, we identified Decorin as a hypertrophy-inducing myokine candidate. Therefore, in this study, we demonstrated that 39 °C mild heat stimulation contributes to muscle hypertrophy through enhancing Decorin gene expression in C2C12 and mouse primary satellite cells.


Assuntos
Temperatura Alta , Músculos , Camundongos , Animais , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Decorina/metabolismo , Hipertrofia/metabolismo , Músculo Esquelético/metabolismo
11.
Biomed Pharmacother ; 155: 113757, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36271545

RESUMO

Colorectal cancer (CRC) progression is strongly influenced by the tumor microenvironment (TME) in which cancer-associated fibroblasts (CAFs) are the major components influencing CRC growth and progression. The present study aimed to investigate the effect of YAP on F-actin arrangement in CAF transformation and the possibility of using YAP as a target for inhibiting CRC growth and progression. Conditioned media were collected from direct interaction between CRC cells and fibroblasts. CAF markers were investigated by flow cytometry, western blot analysis, and immunofluorescence assay in CM-treated fibroblasts. Promoting the CRC progression of conditioned media was determined in CRC cells by using MTT assay, fluorescence assay, wound healing assay, transwell migration assay, and tubulogenesis. The results showed that the conditioned media induced the expression of CAF markers associated with the central rearrangement of F-actin in colon fibroblasts, upregulating and promoting the nuclear translocation of YAP. The conditioned media also significantly promoted the proliferation, migration, invasion, and angiogenesis of CRC cells. Interestingly, Verteporfin, a YAP inhibitor during cocultivation, abolished the conversion of CAFs and inhibited proliferation, migration, invasion, and angiogenesis in CRC cells. Moreover, bioinformatics analysis was employed to determine the potential role of YAP as a prognostic marker in CRC patients from databases. The results suggested that YAP has higher expression in CRC patients and is associated with a poor prognosis. In conclusion, these findings demonstrate that YAP-related F-actin rearrangement may be a potential new target of combination therapy with a focus on targeting TME.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos Associados a Câncer/metabolismo , Actinas/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Verteporfina/farmacologia , Verteporfina/metabolismo , Neoplasias Colorretais/patologia , Proliferação de Células , Movimento Celular , Linhagem Celular Tumoral , Microambiente Tumoral
12.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232829

RESUMO

Hepatic metastasis is the critical factor determining tumor-associated mortality in different types of cancer. This is particularly true for uveal melanoma (UM), which almost exclusively metastasizes to the liver. Hepatic stellate cells (HSCs) are the precursors of tumor-associated fibroblasts and support the growth of metastases. However, the underlying mechanisms are widely unknown. Fibroblast growth factor (FGF) signaling is dysregulated in many types of cancer. The aim of this study was to analyze the pro-tumorigenic effects of HSCs on UM cells and the role of FGFs in this crosstalk. Conditioned medium (CM) from activated human HSCs significantly induced proliferation together with enhanced ERK and JNK activation in UM cells. An in silico database analysis revealed that there are almost no mutations of FGF receptors (FGFR) in UM. However, a high FGFR expression was found to be associated with poor survival for UM patients. In vitro, the pro-tumorigenic effects of HSC-CM on UM cells were abrogated by a pharmacological inhibitor (BGJ398) of FGFR1/2/3. The expression analysis revealed that the majority of paracrine FGFs are expressed by HSCs, but not by UM cells, including FGF9. Furthermore, the immunofluorescence analysis indicated HSCs as a cellular source of FGF9 in hepatic metastases of UM patients. Treatment with recombinant FGF9 significantly enhanced the proliferation of UM cells, and this effect was efficiently blocked by the FGFR1/2/3 inhibitor BGJ398. Our study indicates that FGF9 released by HSCs promotes the tumorigenicity of UM cells, and thus suggests FGF9 as a promising therapeutic target in hepatic metastasis.


Assuntos
Neoplasias Hepáticas , Neoplasias Uveais , Proliferação de Células , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Melanoma , Compostos de Fenilureia , Pirimidinas , Neoplasias Uveais/metabolismo
13.
Cells ; 11(20)2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36291089

RESUMO

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration.


Assuntos
Meios de Cultivo Condicionados , Polpa Dentária , Células Endoteliais da Veia Umbilical Humana , Células-Tronco Mesenquimais , Osteoblastos , Dente Decíduo , Animais , Humanos , Camundongos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Polpa Dentária/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Dente Decíduo/citologia
14.
FASEB J ; 36(11): e22597, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36197688

RESUMO

Estrogen signaling has been extensively studied, especially in cancers that express estrogen receptor alpha (ERα). However, little is known regarding the effect of estrogen on cancer-associated fibroblasts (CAFs). Here, we explored the role of estrogen signaling of CAFs in gastric cancer (GC) progression. We investigated the phenotypic changes in CAFs upon 17ß-estradiol (E2) treatment using ERα-negative/positive CAFs, and the conditioned media (CM) collected from these were compared with regard to cancer cell proliferation, migration, and invasion. A paracrine factor was found using a cytokine array and was confirmed using qRT-PCR, western blotting, and enzyme-linked immunosorbent assays. ERα-CD147-matrix metalloproteinase (MMP) axis was confirmed by knockdown experiments using specific siRNAs. We found that a subset of CAFs expressed ERα. ERα-positive CAFs were responsive to E2, inducing ERα expression in a dose-dependent manner. Although E2 did not induce the proliferation of ERα-positive CAFs, the CM from E2-bound ERα-positive CAFs significantly promoted cancer cell migration and invasion. Cytokine array revealed that CD147 was induced in ERα-positive CAFs upon E2 treatment; this was mediated via ERα. Increased CD147 upregulated MMP2 and MMP9 in CAFs, and also influenced cancer cells in a paracrine manner to increase MMPs and CD147 in cancer cells. High CD147 expression in tumor tissue was associated with a worse prognosis in GC patients. Our data suggest that estrogen signaling activation in CAFs and the byproduct CD147 are among the critical mediators between the interplay of CAFs and cancer cells to facilitate cancer progression.


Assuntos
Basigina/metabolismo , Fibroblastos Associados a Câncer , Neoplasias Gástricas , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Estrogênios/farmacologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Gástricas/patologia
15.
Oxid Med Cell Longev ; 2022: 4320809, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246404

RESUMO

Background: Cancer-associated fibroblasts (CAFs) within the tumor microenvironment are key players in tumorigenesis and tumor development. Nevertheless, the regulatory mechanisms of CAFs on lung squamous cell carcinoma- (LUSC-) associated remain poorly elucidated. Methods: The microarray dataset GSE22874, containing 30 specimens of primary culture of normal fibroblasts (NFs) and 8 specimens of cancer-associated fibroblasts (CAFs) samples derived from LUSC, was retrieved from the Gene Expression Omnibus (GEO) database and then calculated by using the R language (limma package) to identify differentially expressed genes (DEGs). CAF-conditioned medium (CAF-CM) was collected and used to culture LUSC cells, followed by assessment of cell proliferation, apoptosis, and oxidative stress levels by using CCK-8, annexin V-FITC/PI double staining and ELISA assays. Subsequently, COL10A1 was knocked down in CAFs to assess the role of COL10A1 in CAF regulation of LUSC behavior. Bioinformatics online analysis and MeRIP were applied to predict and test the m6A modification of COL10A1 mRNA and the regulatory relationship with METTL3. Rescue experiments were next performed to explore the effects of METTL3 and COL10A1 in CAFs on LUSC cell proliferation, apoptosis, and oxidative stress. LUSC tumor cells with or without (COL10A1-silenced) CAFs were subcutaneously inoculated in nude mice to evaluate the effect of COL10A1 in CAFs on LUSC tumor growth. Results: Elevated expression of COL10A1 was found in LUSC-derived CAFs by GSE22874 dataset analysis. We discovered that COL10A1 and METTL3 was expressed in both LUSC cells and matched CAFs, while COL10A1 expression was prominently higher in CAFs than in LUSC cells. CAF-CM memorably encouraged LUSC cell proliferation and suppressed apoptosis-induced oxidative stress, which was reversed by interfering with COL10A1 expression in CAFs, suggesting that COL10A1 might be secreted by CAFs into the culture medium to exert its effects inside LUSC cells. Global m6A modification was decreased in METTL3 knocked down CAFs. M6A modification, expression levels, and stability of COL10A1 mRNA were impaired upon METTL3 knockdown in CAFs. Overexpression of COL10A1 in CAFs partially reversed the effect of METTL3 knockdown on the malignant behavior of LUSC cells. In vivo studies confirmed that CAFs accelerated LUSC tumor growth, and this effect was counteracted by COL10A1 silencing. Conclusions: COL10A1 secreted by CAFs could facilitate LUSC cell proliferation and repress apoptosis-induced oxidative stress, and the mechanism was due to elevated expression mediated by METTL3 promoting its mRNA m6A modification, thereby accelerating tumor growth.


Assuntos
Fibroblastos Associados a Câncer , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animais , Apoptose/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Colágeno Tipo X , Meios de Cultivo Condicionados/metabolismo , Fibroblastos/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Metilação , Metiltransferases , Camundongos , Camundongos Nus , Estresse Oxidativo , RNA Mensageiro/metabolismo , Sincalida/metabolismo
16.
Sci Rep ; 12(1): 16656, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36198906

RESUMO

Runt-related transcription factor 2 (Runx2), a regulator of osteoblast differentiation, is pathologically involved in vascular calcification; however, the significance of Runx2 in cardiac homeostasis remains unclear. Here, we investigated the roles of Runx2 in cardiac remodeling after myocardial infarction (MI). The expression of Runx2 mRNA and protein was upregulated in murine hearts after MI. Runx2 was expressed in heart-infiltrating myeloid cells, especially in macrophages, at the border zone of post-infarct myocardium. To analyze the biological functions of Runx2 in cardiac remodeling, myeloid cell-specific Runx2 deficient (CKO) mice were exposed to MI. After MI, ventricular weight/tibia length ratio was increased in CKO mice, concomitant with severe cardiac dysfunction. Cardiac fibrosis was exacerbated in CKO mice, consistent with the upregulation of collagen 1a1 expression. Mechanistically, immunohistochemical analysis using anti-CD31 antibody showed that capillary density was decreased in CKO mice. Additionally, conditioned culture media of myeloid cells from Runx2 deficient mice exposed to MI induced the tube formation of vascular endothelial cells to a lesser extent than those from control mice. RNA-sequence showed that the expression of pro-angiogenic or anti-angiogenic factors was altered in macrophages from Runx2-deficient mice. Collectively, Runx2+ myeloid cells infiltrate into post-infarct myocardium and prevent adverse cardiac remodeling, at least partially, by regulating endothelial cell function.


Assuntos
Infarto do Miocárdio , Remodelação Ventricular , Animais , Colágeno/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Remodelação Ventricular/genética
17.
Bull Exp Biol Med ; 173(5): 633-635, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36210409

RESUMO

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).


Assuntos
Medula Óssea , Eritropoetina , Animais , Medula Óssea/metabolismo , Células da Medula Óssea , Meios de Cultivo Condicionados/metabolismo , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Glucose/metabolismo , Humanos , Lactatos/metabolismo , Camundongos , NADP/metabolismo , Oxirredutases , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Baço
18.
Sci Rep ; 12(1): 17367, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253398

RESUMO

Synovium is critical for maintaining joint homeostasis and may contribute to mechanobiological responses during joint movement. We investigated mechanobiological responses of whole synovium from patients with late-stage knee osteoarthritis (OA). Synovium samples were collected during total knee arthroplasty and assigned to histopathology or cyclic 10% tensile strain loading, including (1) static (control); (2) low-frequency (0.3 Hz); and iii) high-frequency (1.0 Hz) for 30-min. After 6-h incubation, tissues were bisected for RNA isolation and immunostaining (3-nitrotyrosine; 3-NT). RNA sequencing was analyzed for differentially expressed genes and pathway enrichment. Cytokines and lactate were measured in conditioned media. Compared to controls, low-frequency strain induced enrichment of pathways related to interferon response, Fc-receptor signaling, and cell metabolism. High-frequency strain induced enrichment of pathways related to NOD-like receptor signaling, high metabolic demand, and redox signaling/stress. Metabolic and redox cell stress was confirmed by increased release of lactate into conditioned media and increased 3-NT formation in the synovial lining. Late-stage OA synovial tissue responses to tensile strain include frequency-dependent increases in inflammatory signaling, metabolism, and redox biology. Based on these findings, we speculate that some synovial mechanobiological responses to strain may be beneficial, but OA likely disturbs synovial homeostasis leading to aberrant responses to mechanical stimuli, which requires further validation.


Assuntos
Osteoartrite do Joelho , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Humanos , Interferons/metabolismo , Lactatos/metabolismo , Proteínas NLR/metabolismo , Osteoartrite do Joelho/patologia , RNA/metabolismo , Membrana Sinovial/metabolismo
19.
Pharm Biol ; 60(1): 2011-2024, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36239618

RESUMO

CONTEXT: Shenlian extract (SL) is a combination of Salvia miltiorrhiza Bge. (Labiatae) and Andrographis paniculata (Burm. F.) Wall. Ex Nees (Acanthaceae) extracts, which promote blood circulation and clear endogenous heat toxins. Myocardial ischaemia-reperfusion injury (MI/RI) is aggravated myocardial tissue damage induced by reperfusion therapy after myocardial infarction. OBJECTIVES: This study explores the effect of SL on MI/RI and the underlying mechanism. MATERIALS AND METHODS: Primary peritoneal macrophages (pMACs) were treated with LPS and SL (5, 10 or 20 µg/mL) for 24 h. The myocardial ischaemia-reperfusion (MI/R) model was established after administration of different doses of SL (90, 180 or 360 mg/kg). Myocardial tissue injury was assessed by methylthiazolyl tetrazolium (TTC) staining and levels of creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in mice. The double immunofluorescence staining of iNOS/F4/80 and CD86/F4/80 was used to detect macrophage M1 polarization. The levels of miR-155, inflammatory factors and chemokines were detected by qRT-PCR or ELISA. CD86, iNOS, SOCS3, JAK2, p-JAK2, STAT3 and p-STAT3 proteins expressions in macrophages were analyzed by western blotting. Conditioned medium transfer systems were designed to unite M1 macrophages with H/R cardiomyocytes, and cell apoptosis was detected by TUNEL staining, western blotting or immunohistochemistry. RESULTS: SL reduced apoptosis, diminished CK and LDH levels, raised SOD concentration and decreased infarct size in the MI/R model. Meanwhile, SL decreased miR-155 level, inhibited M1 macrophage polarization and inflammation. Furthermore, SL promoted SOCS3 expression and blocked JAK2/STAT3 pathway in vitro. CONCLUSIONS: SL may be a promising TCM candidate for MI/RI. The underlying mechanisms could be associated with inhibition of M1 macrophage polarization via down-regulating miR-155.


Assuntos
MicroRNAs , Traumatismo por Reperfusão Miocárdica , Animais , Apoptose , Creatina Quinase/metabolismo , Creatina Quinase/farmacologia , Creatina Quinase/uso terapêutico , Meios de Cultivo Condicionados/metabolismo , Lactato Desidrogenases/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Superóxido Dismutase/metabolismo
20.
Cells ; 11(19)2022 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-36230929

RESUMO

Breast cancer (BC) cell secretome in the tumor microenvironment (TME) facilitates neo-angiogenesis by promoting vascular endothelial cell (VEC) growth. Drugs that block BC cell growth or angiogenesis can restrict tumor growth and are of clinical relevance. Molecules that can target both BC cell and VEC growth as well as BC secretome may be more effective in treating BC. Since small non-coding microRNAs (miRs) regulate cell growth and miR193a-3p has onco-suppressor activity, we investigated whether miR193a-3p inhibits MCF-7-driven growth (proliferation, migration, capillary formation, signal transduction) of VECs. Using BC cells and VECs grown in monolayers or 3D spheroids and gene microarrays, we demonstrate that: pro-growth effects of MCF-7 and MDA-MB231 conditioned medium (CM) are lost in CM collected from MCF-7/MDA-MB231 cells pre-transfected with miR193a-3p (miR193a-CM). Moreover, miR193a-CM inhibited MAPK and Akt phosphorylation in VECs. In microarray gene expression studies, miR193a-CM upregulated 553 genes and downregulated 543 genes in VECs. Transcriptomic and pathway enrichment analysis of differentially regulated genes revealed downregulation of interferon-associated genes and pathways that induce angiogenesis and BC/tumor growth. An angiogenesis proteome array confirmed the downregulation of 20 pro-angiogenesis proteins by miR193a-CM in VECs. Additionally, in MCF-7 cells and VECs, estradiol (E2) downregulated miR193a-3p expression and induced growth. Ectopic expression of miR193a-3p abrogated the growth stimulatory effects of estradiol E2 and serum in MCF-7 cells and VECs, as well as in MCF-7 and MCF-7+VEC 3D spheroids. Immunostaining of MCF-7+VEC spheroid sections with ki67 showed miR193a-3p inhibits cell proliferation. Taken together, our findings provide first evidence that miR193a-3p abrogates MCF-7-driven growth of VECs by altering MCF-7 secretome and downregulating pro-growth interferon signals and proangiogenic proteins. Additionally, miR193a-3p inhibits serum and E2-induced growth of MCF-7, VECs, and MCF-7+VEC spheroids. In conclusion, miRNA193a-3p can potentially target/inhibit BC tumor angiogenesis via a dual mechanism: (1) altering proangiogenic BC secretome/TME and (2) inhibiting VEC growth. It may represent a therapeutic molecule to target breast tumor growth.


Assuntos
Neoplasias da Mama , MicroRNAs , Neoplasias da Mama/patologia , Proliferação de Células/genética , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Estradiol/metabolismo , Feminino , Humanos , Interferons/metabolismo , Antígeno Ki-67/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Secretoma , Transcriptoma/genética , Microambiente Tumoral
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