RESUMO
The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.
Assuntos
Fator 7 de Crescimento de Fibroblastos , Queratinócitos , Alicerces Teciduais , Animais , Camundongos , Alicerces Teciduais/química , Queratinócitos/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/química , Fator 7 de Crescimento de Fibroblastos/farmacologia , Porosidade , Peptídeos/química , Peptídeos/farmacologia , Cicatrização/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Polímeros/química , Polímeros/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Masculino , Regeneração/efeitos dos fármacos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Pé Diabético/tratamento farmacológico , Pé Diabético/patologia , Pé Diabético/terapia , HumanosRESUMO
BACKGROUND: Diabetic cataract (DC) is a common complication of diabetes and its etiology and progression are multi-factorial. In this study, the roles of specific protein 1 (SP1) and fibroblast growth factor 7 (FGF7) in DC development were explored. METHODS: DC cell model was established by treating SRA01/04 cells with high glucose (HG). MTT assay was conducted to evaluate cell viability. Transwell assay and wound-healing assay were performed to assess cell migration and invasion. Western blot assay and qRT-PCR assay were conducted to measure the expression of N-cadherin, E-cadherin, Collagen I, Fibronectin, SP1 and FGF7 expression. CHIP assay and dual-luciferase reporter assay were conducted to analyze the combination between FGF7 and SP1. RESULTS: FGF7 was upregulated in DC patients and HG-induced SRA01/04 cells. HG treatment promoted SRA01/04 cell viability, migration, invasion and epithelial-mesenchymal transition (EMT), while FGF7 knockdown abated the effects. Transcription factor SP1 activated the transcription level of FGF7 and SP1 overexpression aggravated HG-induced SRA01/04 cell injury. SP1 silencing repressed HG-induced SRA01/04 cell viability, migration, invasion and EMT, but these effects were ameliorated by upregulating FGF7. Additionally, SP1 knockdown inhibited the PI3K/AKT pathway by regulating the transcription level of FGF7. CONCLUSION: Transcription factor SP1 activated the transcription level of FGF7 and the PI3K/AKT pathway to regulate HG-induced SRA01/04 cell viability, migration, invasion and EMT.
Assuntos
Movimento Celular , Sobrevivência Celular , Células Epiteliais , Transição Epitelial-Mesenquimal , Fator 7 de Crescimento de Fibroblastos , Glucose , Cristalino , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fator de Transcrição Sp1 , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Glucose/farmacologia , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Cristalino/metabolismo , Cristalino/citologia , Catarata/metabolismo , Células Cultivadas , Regulação da Expressão GênicaRESUMO
BACKGROUND: Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood. METHODS: Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 µM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay. RESULTS: BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo. CONCLUSION: BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.
Assuntos
Berberina , Neoplasias da Mama , Proliferação de Células , Fator 7 de Crescimento de Fibroblastos , Metiltransferases , RNA Mensageiro , Humanos , Berberina/farmacologia , Berberina/uso terapêutico , Berberina/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Feminino , Camundongos , Proliferação de Células/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator 7 de Crescimento de Fibroblastos/genética , Apoptose/efeitos dos fármacos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Nus , Movimento Celular/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Muscle satellite cells (MuSCs) exert essential roles in skeletal muscle adaptation to growth, injury and ageing, and their functions are extensively modulated by microenvironmental factors. However, the current knowledge about the interaction of MuSCs with niche cells is quite limited. METHODS: A 10× single-cell RNA sequencing (scRNA-seq) was performed on porcine longissimus dorsi and soleus (SOL) muscles to generate a single-cell transcriptomic dataset of myogenic cells and other cell types. Sophisticated bioinformatic analyses, including unsupervised clustering analysis, marker gene, gene set variation analysis (GSVA), AUCell, pseudotime analysis and RNA velocity analysis, were performed to explore the heterogeneity of myogenic cells. CellChat analysis was used to demonstrate cell-cell communications across myogenic cell subpopulations and niche cells, especially fibro-adipogenic progenitors (FAPs). Integrated analysis with human and mice datasets was performed to verify the expression of FGF7 across diverse species. The role of FGF7 on MuSC proliferation was evaluated through administering recombinant FGF7 to porcine MuSCs, C2C12, cardiotoxin (CTX)-injured muscle and d-galactose (d-gal)-induced ageing model. RESULTS: ScRNA-seq totally figured out five cell types including myo-lineage cells and FAPs, and myo-lineage cells were further classified into six subpopulations, termed as RCN3+, S100A4+, ID3+, cycling (MKI67+), MYF6+ and MYMK+ satellite cells, respectively. There was a higher proportion of cycling and MYF6+ cells in the SOL population. CellChat analysis uncovered a particular impact of FAPs on myogenic cells mediated by FGF7, which was relatively highly expressed in SOL samples. Administration of FGF7 (10 ng/mL) significantly increased the proportion of EdU+ porcine MuSCs and C2C12 by 4.03 ± 0.81% (P < 0.01) and 6.87 ± 2.17% (P < 0.05), respectively, and knockdown of FGFR2 dramatically abolished the pro-proliferating effects (P < 0.05). In CTX-injured muscle, FGF7 significantly increased the ratio of EdU+/Pax7+ cells by 15.68 ± 5.45% (P < 0.05) and elevated the number of eMyHC+ regenerating myofibres by 19.7 ± 4.25% (P < 0.01). Under d-gal stimuli, FGF7 significantly reduced γH2AX+ cells by 17.19 ± 3.05% (P < 0.01) in porcine MuSCs, induced EdU+ cells by 4.34 ± 1.54% (P < 0.05) in C2C12, and restored myofibre size loss and running exhaustion in vivo (all P < 0.05). CONCLUSIONS: Our scRNA-seq reveals a novel interaction between muscle FAPs and satellite cells mediated by FGF7-FGFR2. Exogenous FGF7 augments the proliferation of satellite cells and thus benefits muscle regeneration and counteracts age-related myopathy.
Assuntos
Fator 7 de Crescimento de Fibroblastos , Células Satélites de Músculo Esquelético , Transdução de Sinais , Animais , Humanos , Camundongos , Adipogenia , Diferenciação Celular , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , RNA-Seq , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Análise da Expressão Gênica de Célula Única/métodos , Células-Tronco/metabolismo , SuínosRESUMO
Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), plays a critical role in embryonic development, cell proliferation, and differentiation. However, efficient production of recombinant KGF remains a challenge due to its low expression levels and high tendency for aggregation in Escherichia coli. This study aimed to enhance the expression and solubility of KGF by employing different protein tags-PDIb'a', MBP, and His-fused to the N-terminus of KGF. Among these, H-PDIb'a'-KGF demonstrated superior stability and was selected for large-scale production and purification. The purified KGF was confirmed through liquid chromatography with tandem mass spectrometry analysis, which showed an 81% fragment mass identification coverage. Biological activity assessments using human breast cancer MCF-7 cells indicated that purified KGF significantly increased cell proliferation, with an EC50 of 6.4 ± 0.5 pM. Interestingly, PDIb'a' alone also exhibited a stimulatory effect on MCF-7 cells. Furthermore, the purified KGF enhanced the wound healing of HaCaT keratinocytes in a dose-dependent manner. These findings provide valuable insights into the efficient production and functional characterization of recombinant KGF for potential applications in therapeutic interventions.
Assuntos
Fator 7 de Crescimento de Fibroblastos , Humanos , Diferenciação Celular , Proliferação de Células , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Queratinócitos/metabolismo , Células MCF-7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Ovarian cancer (OC) is distinguished by its aggressive nature and the limited efficacy of current treatment strategies. Recent studies have emphasized the significant role of cancer-associated fibroblasts (CAFs) in OC development and progression. METHODS: Employing sophisticated machine learning techniques on bulk transcriptomic datasets, we identified fibroblast growth factor 7 (FGF7), derived from CAFs, as a potential oncogenic factor. We investigated the relationship between FGF7 expression and various clinical parameters. A series of in vitro experiments were undertaken to evaluate the effect of CAFs-derived FGF7 on OC cell activities, such as proliferation, migration, and invasion. Single-cell transcriptomic analysis was also conducted to elucidate the interaction between FGF7 and its receptor. Detailed mechanistic investigations sought to clarify the pathways through which FGF7 fosters OC progression. RESULTS: Our findings indicate that higher FGF7 levels correlate with advanced tumor stages, increased vascular invasion, and poorer prognosis. CAFs-derived FGF7 significantly enhanced OC cell proliferation, migration, and invasion. Single-cell analysis and in vitro studies revealed that CAFs-derived FGF7 inhibits the ubiquitination and degradation of hypoxia-inducible factor 1 alpha (HIF-1α) via FGFR2 interaction. Activation of the FGF7/HIF-1α pathway resulted in the upregulation of mesenchymal markers and downregulation of epithelial markers. Importantly, in vivo treatment with neutralizing antibodies targeting CAFs-derived FGF7 substantially reduced tumor growth. CONCLUSION: Neutralizing FGF7 in the medium or inhibiting HIF-1α signaling reversed the effects of FGF7-mediated EMT, emphasizing the dependence of FGF7-mediated EMT on HIF-1α activation. These findings suggest that targeting the FGF7/HIF-1α/EMT axis may offer new therapeutic opportunities to intervene in OC progression.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Ovarianas , Humanos , Feminino , Fibroblastos Associados a Câncer/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Linhagem Celular Tumoral , Transdução de Sinais , Neoplasias Ovarianas/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transição Epitelial-Mesenquimal/genética , Movimento Celular/genéticaRESUMO
Oral mucositis (OM) is a severe complication of cancer therapies caused by off-target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM-OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane-lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM-OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32-36h. Presence of KGF in OM-OC protected tissues from damage caused by cancer treatments in a dose-dependent manner, being more effective at 10 ng/mL. As verified by F-actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM-OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL-8 and TNF-a expression at Day 12 and 18, and TGF-ß1 at Day 18 in OM-OC. Taken together, these findings support the utility of OM-OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.
Assuntos
Fator 7 de Crescimento de Fibroblastos , Queratinócitos , Proteínas Recombinantes , Estomatite , Humanos , Estomatite/tratamento farmacológico , Estomatite/patologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Proteínas Recombinantes/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Cisplatino/farmacologia , Neoplasias/tratamento farmacológico , Gengiva , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action. METHODS: Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and ß-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging. RESULTS: We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of ß-catenin, and accelerated ß-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone. CONCLUSIONS: ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.
Assuntos
Lesão Pulmonar Aguda , Isoxazóis , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Tiofenos , Humanos , beta Catenina/metabolismo , beta Catenina/farmacologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Lesão Pulmonar Aguda/terapia , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
PURPOSE: To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM). METHODS: A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed. RESULTS: RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor ß (TGF-ß) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-ß concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-ß concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT. CONCLUSIONS: HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-ß and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.
Assuntos
Fator de Crescimento Epidérmico , Fotoquimioterapia , Humanos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Rosa Bengala/farmacologia , Células Cultivadas , Fibroblastos/metabolismo , Movimento Celular , Fator de Crescimento Transformador beta/metabolismo , Células Epiteliais , Caderinas/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologiaRESUMO
Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.
Assuntos
Lesão Pulmonar Aguda , Células-Tronco Mesenquimais , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Canais Epiteliais de Sódio/metabolismo , NF-kappa B/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Células-Tronco Mesenquimais/metabolismo , PulmãoRESUMO
The objective of this study was to investigate the potential effects of a formulation derived from the bioactive fraction of nanostructured Bacopa procumbens (BFNB) on the promotion of hair growth in C57BL/6 mice. The characterization of the follicular phases and histomorphological analysis showed that the topical application of the formulation for 15 days significantly increased pigmentation and hair growth on the dorsum and head of the mice. Additionally, an acceleration of the follicular cycle phases was observed, along with an increase in the number of follicles, hair length, and diameter, compared to mice treated with minoxidil. In silico analysis and molecular characterization demonstrated that BFNB enhances the expression of epidermal growth factor (EGF) and fibroblast growth factor 7 (FGF7), activating the PI3K-AKT-ß-catenin signaling pathway, as well as the expression of PCNA, KI-67, Cyclin D1, and Cyclin E, regulating the cell cycle and cell proliferation, crucial events for hair regeneration. Our results strongly suggest the utility of BFNB as a therapeutic alternative to stimulate hair growth and promote hair health.
Assuntos
Fator de Crescimento Epidérmico , beta Catenina , Animais , Camundongos , beta Catenina/metabolismo , Cateninas/metabolismo , Proliferação de Células , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Cabelo/metabolismo , Folículo Piloso/metabolismo , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Ulcerative colitis (UC) is a chronic recurrent disease affecting the gastrointestinal tract especially colorectum. Keratinocyte growth factor (KGF) plays the vital roles in maintaining the colonic mucosal barrier. The poor stability and off-target of KGF were two hindering factors for its clinical application. Herein, in situ hydrogel (PE) with mucoadhesive ability was constructed by using temperature-sensitive poloxamer and EGCG as hydrogel-forming material and adhesive enhancer, respectively. Incorporation of EGCG led to the slight decrease of the gelled temperature and shortened the gelled time of PE hydrogel. When the concentration of EGCG is 0.1 %, PE hydrogel exhibits the suitable viscosity of 280 ± 20 Pa·s and the strong adhesive force of 725 ± 25 mN. KGF was soluble in cold PE solution to obtain KGF-loaded PE hydrogel (KGF@PE). PE hydrogel could improve the stability of KGF in vitro. KGF@PE not only could recover greatly the body weight of TNBS-induced rats but also repair their colonic morphology and goblet cell function. Moreover, the potential of repairing the epithelial barrier was indicated by upregulating tight junction proteins. Importantly, the safety of KGF@PE hydrogel for colitis was also confirmed on AOM/DSS-induced mice models. Conclusively, KGF@PE may be a promising therapeutic platform without obvious side effect for ulcerative colitis.
Assuntos
Colite Ulcerativa , Colite , Ratos , Camundongos , Animais , Colite Ulcerativa/tratamento farmacológico , Hidrogéis/farmacologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Adesivos/farmacologia , Colo/metabolismo , Modelos Animais de Doenças , Sulfato de Dextrana/efeitos adversos , Mucosa Intestinal/metabolismo , Colite/metabolismoRESUMO
Ovarian granulosa cells (OGCs) play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on traditional 2D cultures. In the present study, we used the hanging drop culture method to culture rat OGCs (rOGCs) and assessed the effects of 3D conditions on their proliferation and gene expression profiles. Compared with those grown in 2D conditions, rOGCs grown in 3D cultures showed a significantly different spatial cell distribution and cell alignment under electron microscopy. In particular, rOGCs in 3D cultures showed abundant rough and microvilli-like structures on their cell surface. Here, we showed that these cells grew slowly following 3D culture; the G0/G1-phase increased and the S- and G2/M-phases decreased. Using whole-transcriptome sequencing analysis, 501 genes were shown to have been significantly upregulated and 502 were shown to have been downregulated. Differentially expressed genes were most enriched in pathways involved in focal adhesion, MAPK, and PI3K/Akt signaling according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Western blotting revealed that SPP1 and FGF7 in the PI3K/Akt pathway were significantly upregulated following 3D culture. These findings improve our understanding of OGCs in real 3D environments in vivo and provide possible avenues for future research on OGCs.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Feminino , Ratos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células da Granulosa , Transdução de Sinais , Transcriptoma , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologiaRESUMO
Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.
Assuntos
Diferenciação Celular , Fator 7 de Crescimento de Fibroblastos , Queratinócitos , Células-Tronco , Humanos , Colágeno , Colágeno Tipo I/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Células HEK293 , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Acute myocardial infarction (MI) triggers oxidative stress, which worsen cardiac function, eventually leads to remodeling and heart failure. Unfortunately, effective therapeutic approaches are lacking. Fibroblast growth factor 7 (FGF7) is proved with respect to its proliferative effects and high expression level during embryonic heart development. However, the regulatory role of FGF7 in cardiovascular disease, especially MI, remains unclear. FGF7 expression was significantly decreased in a mouse model at 7 days after MI. Further experiments suggested that FGF7 alleviated MI-induced cell apoptosis and improved cardiac function. Mechanistic studies revealed that FGF7 attenuated MI by inhibiting oxidative stress. Overexpression of FGF7 actives nuclear factor erythroid 2-related factor 2 (Nrf2) and scavenging of reactive oxygen species (ROS), and thereby improved oxidative stress, mainly controlled by the phosphatidylinositol-3-kinase α (PI3Kα)/AKT signaling pathway. The effects of FGF7 were partly abrogated in Nrf2 deficiency mice. In addition, overexpression of FGF7 promoted hexokinase2 (HXK2) and mitochondrial membrane translocation and suppressed mitochondrial superoxide production to decrease oxidative stress. The role of HXK2 in FGF7-mediated improvement of mitochondrial superoxide production and protection against MI was verified using a HXK2 inhibitor (3-BrPA) and a HXKII VDAC binding domain (HXK2VBD) peptide, which competitively inhibits localization of HXK2 on mitochondria. Furthermore, inhibition of PI3Kα/AKT signaling abolished regulation of Nrf2 and HXK2 by FGF7 upon MI. Together, these results indicate that the cardio protection of FGF7 under MI injury is mostly attributable to its role in maintaining redox homeostasis via Nrf2 and HXK2, which is mediated by PI3Kα/AKT signaling.
Assuntos
Infarto do Miocárdio , Fator 2 Relacionado a NF-E2 , Animais , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , SuperóxidosRESUMO
PURPOSE: Diabetic retinopathy (DR), the major complication of diabetes, is the leading cause of vision loss and blindness globally. Altered circular RNAs (circRNAs) expression has been found to be involved in DR process. Hence, this work aimed to explore the role and mechanism of circCOL1A2 in DR. METHODS: Human retinal microvascular endothelial cells (RMECs) treated with high glucose (HG) were used for functional analysis. Levels of genes and proteins were detected using quantitative real-time polymerase chain reaction and western blotting. In vitro experiments were conducted by transwell, tube formation, CCK-8 assays and ELISA, respectively. The binding interaction between miR-646 and circCOL1A2 or FGF7 (Fibroblast Growth Factor 7) was confirmed using dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: CircCOL1A2 was highly expressed in retinal tissues of DR patients and HG-induced RMECs. Then RMECs were exposed to HG treatment to mimic the diabetic conditions in vitro. Functionally, circCOL1A2 knockdown attenuated HG-evoked RMEC migration, proliferation, angiogenesis, blood-retina barrier (BRB) injury and inflammation. Mechanistically, circCOL1A2 functioned as a sponge for miR-646, and miR-646 directly targeted FGF7. Further rescue experiments showed that miR-646 inhibition abated the protective effects of circCOL1A2 knockdown on RMEC function under HG treatment. Besides that, miR-646 was decreased in HG-induced RMECs, re-expression of miR-646 reversed HG-evoked RMEC dysfunction, which was rescued by FGF7 overexpression. CONCLUSION: CircCOL1A2 silencing can suppress HG-induced migration, proliferation, angiogenesis, BRB injury and inflammation in RMECs through miR-646/FGF7 axis, suggesting the potential involvement of circCOL1A2 in DR process.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , MicroRNAs , Proliferação de Células , Retinopatia Diabética/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Glucose/toxicidade , Humanos , Inflamação/metabolismo , MicroRNAs/genética , Neovascularização Patológica/metabolismo , RNA Circular , Retina/metabolismo , Sincalida/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by Amyloid-ß accumulation-induced neuronal injury. Emerging evidence shows that circular RNA (circRNA) is involved in AD development. The aim of this study was to illustrate the role of circ-HUWE1 in Amyloid-ß accumulation-induced neuronal injury. Quantitative real-time PCR (qPCR) or western blot was conducted for the expression analysis of circ-HUWE1, miR-433-3p, and fibroblast growth factor 7 (FGF7). In functional assays, cell viability was determined by CCK-8 assay, and cell apoptosis was examined by flow cytometry assay, the protein levels of apoptosis-related markers, and caspase1 or caspase3 activity. The release of pro-inflammatory factors was monitored by ELISA. The predicted binding relationship between miR-433-3p and circ-HUWE1 or FGF7 was validated by dual-luciferase reporter assay. We discovered that circ-HUWE1 absence alleviated Amyloid-ß-induced cell viability degradation, cell apoptosis, and inflammatory responses in SK-N-SH cells. MiR-433-3p was a target of circ-HUWE1, and miR-433-3p inhibition reversed the effects of circ-HUWE1 knockdown. In addition, FGF7 was a downstream target of miR-433-3p whose function could be abolished by FGF7 reintroduction. Circ-HUWE1 positively regulated FGF7 expression via competitively targeting miR-433-3p. Moreover, circ-HUWE1 knockdown activated the WNT signaling pathway in Amyloid-ß-treated SK-N-SH cells by targeting the miR-433-3p/FGF7 axis. In conclusion, circ-HUWE1 knockdown alleviates Amyloid-ß-induced neuronal injury in SK-N-SH cells via miR-433-3p release-mediated FGF7 depletion.
Assuntos
Doença de Alzheimer , MicroRNAs , Doenças Neurodegenerativas , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apoptose , Linhagem Celular Tumoral , Regulação para Baixo , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and ß1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).
Assuntos
Fator 7 de Crescimento de Fibroblastos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Epitélio , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Granulócitos/metabolismo , Humanos , Queratinócitos , Macrófagos/metabolismoRESUMO
Although full-length fibroblast growth factor 7 (FGF7) blocks cyclophosphamide-induced urothelial apoptosis in mice, limitations include high production costs because of its large size. We previously identified a small peptide derived from FGF2 that mitigated acute radiation syndrome as well as full-length FGF2. Based on the sequence of the FGF2 peptide, we synthesized a corresponding 19 amino acid FGF7 peptide (FGF7p). Our objectives were to determine if systemic FGF7p triggered the downstream targets and protected against cyclophosphamide bladder injury similar to full-length FGF7. We administered FGF7p or vehicle subcutaneously (SQ) to mice subjected to no injury or intraperitoneal (IP) cyclophosphamide and harvested bladders 1 day after injury. We then performed hematoxylin and eosin, TUNEL and immunofluorescence (IF) staining. In uninjured mice, a 20 mg/kg threshold FGF7p dose induced expression of phosphorylated (activated) FRS2α (pFRS2α), and pAKT in urothelium (consistent with cytoprotective effects of FGF7). We then gave FGF7p (20 mg/kg) or vehicle at 72 and 48 h prior to cyclophosphamide. One day after injury, TUNEL staining revealed many more apoptotic urothelial cells with vehicle treatment versus FGF7p treatment. IF for pAKT and readouts of two anti-apoptotic AKT targets (BAD and mTORC1) revealed minimal staining with vehicle treatment, but strong urothelial expression for all markers with FGF7p treatment. In conclusion, FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, similar to FGF7. FGF7p is much more inexpensive to make and has a longer shelf life and higher purity than FGF7.
Assuntos
Bexiga Urinária , Urotélio , Animais , Ciclofosfamida/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/farmacologia , Camundongos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bexiga Urinária/metabolismo , Urotélio/metabolismoRESUMO
Keratinocyte growth factor (KGF) drives phosphorylated (activated) AKT (pAKT) in bladder urothelium, which correlates with cytoprotection from cyclophosphamide. The current study determined whether: i) KGF modifies AKT targets [B-cell lymphoma protein 2-associated agonist of cell death (BAD) and mammalian target of rapamycin complex (mTORC)-1] that could block apoptosis; ii) AKT signaling is required for KGF cytoprotection; iii) direct AKT activation drives cytoprotection; iv) co-administration of KGF and an AKT inhibitor blocks urothelial cytoprotection and AKT and AKT-target activation; and v) an AKT agonist prevents cyclophosphamide-induced urothelial apoptosis. Mice were given KGF and cyclophosphamide (or sham injury), and pBAD (readout of BAD inhibition) or p-p70S6k (pS6, readout of mTORC1 signaling) was assessed. KGF induced pBAD urothelial staining and prevented cyclophosphamide-induced loss of urothelial pS6 staining (likely stabilizing mTORC1 activity). Co-administration of KGF and AKT inhibitor blocked KGF-driven urothelial cytoprotection from cyclophosphamide and prevented pAKT, pBAD, and pS6 urothelial expression. Conversely, systemic AKT agonist blocked cyclophosphamide-induced urothelial apoptosis and induced pAKT, pBAD, and pS6, similar to KGF. Thus, the KGF-AKT signaling axis appeared to phosphorylate (suppress) BAD and prevent cyclophosphamide-induced loss of mTORC1 signaling, both of which likely suppress apoptosis. Additionally, AKT signaling was required for KGF-driven cytoprotection, and direct AKT activation was sufficient for blocking apoptosis. Thus, AKT may be a therapeutic target for blocking urothelial apoptosis from cyclophosphamide.