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1.
J Vis Exp ; (204)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38436407

RESUMO

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.


Assuntos
Comunicação Celular , Organoides , Humanos , Camundongos , Animais , Células NIH 3T3 , Ritmo Circadiano , Fibroblastos
2.
Autoimmunity ; 57(1): 2201412, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38425093

RESUMO

OBJECTIVE: To explore the effect of CD5-like molecule (CD5L) on rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS) and the relative molecular mechanism of CD5L in it. METHODS: Recombinant protein CD5L was used to stimulate the cultured RA-FLS cells. The inflammation-related cytokines were determined by real time-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The signal molecules and apoptosis-related molecules were detected by western blot assay (WB), and cell counting kit-8 (CCK-8) was used to detect the proliferation. RESULTS: CD5L can increase the production of IL-6, IL-8, and TNF-α and this effect can be inhibited by signal pathway inhibitor. At the same time, CD5L activated ERK1/2 MAPK signal, inhibitor treatment can weaken the intensity of phosphorylation. In addition, CD5L can enhance the proliferation ability of RA-FLS. CONCLUSION: CD5L induces the production of inflammatory cytokines in RA-FLS through the ERK1/2 MAPK pathway and increases cell survival.


Assuntos
Artrite Reumatoide , Membrana Sinovial , Humanos , Membrana Sinovial/metabolismo , Sistema de Sinalização das MAP Quinases , Artrite Reumatoide/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Proliferação de Células , Proteínas Reguladoras de Apoptose , Receptores Depuradores/metabolismo
3.
Front Endocrinol (Lausanne) ; 15: 1350958, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469138

RESUMO

With the development of social population ageing, bone fracture has become a global public health problem due to its high morbidity, disability and mortality. Fracture healing is a complex phenomenon involving the coordinated participation of immigration, differentiation and proliferation of inflammatory cells, angioblasts, fibroblasts, chondroblasts and osteoblasts which synthesize and release bioactive substances of extracellular matrix components, Mortality caused by age-related bone fractures or osteoporosis is steadily increasing worldwide as the population ages. Fibroblasts play an important role in the process of fracture healing. However, it is not clear how the growth factors and extracellular matrix stiffness of the bone-regeneration microenvironment affects the function of osteoblasts and fibroblasts in healing process. Therefore, this article focuses on the role of fibroblasts in the process of fracture healing and mechanisms of research progress.


Assuntos
Fraturas Ósseas , Osteoporose , Humanos , Consolidação da Fratura , Regeneração Óssea , Fibroblastos
4.
Hepatol Commun ; 8(4)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38466884

RESUMO

BACKGROUND: Hepatic fibrosis is a progressive disease, which is reversible in the early stages. The current monitoring methods have notable limitations that pose a challenge to early detection. In this study, we evaluated the utility of [18F]AlF-ND-bisFAPI positron emission tomography imaging of fibroblast activation protein (FAP) to monitor the progression of liver fibrosis. METHODS: Two mouse models of liver fibrosis were established by bile duct ligation and carbon tetrachloride administration, respectively. Positron emission tomography imaging was performed with the FAP-specific radiotracer [18F]AlF-ND-bisFAPI for the evaluation of rat HSCs and mouse models of fibrosis and combined with histopathology, immunohistochemical staining, and immunoblotting to elucidate the relationships among radioactivity uptake, FAP levels, and liver fibrosis progression. Furthermore, [18F]AlF-ND-bisFAPI autoradiography was performed to assess tracer binding in liver sections from patients with varying degrees of liver fibrosis. RESULTS: Cell experiments demonstrated that [18F]AlF-ND-bisFAPI uptake was specific in activated HSCs. Compared with control mice, [18F]AlF-ND-bisFAPI uptake in livers increased in the early stages of fibrosis and increased significantly further with disease progression. Immunohistochemistry and western blot analyses demonstrated that FAP expression increased with fibrosis severity. In accordance with the findings in animal models, ex vivo autoradiography on human fibrotic liver sections showed that radioactivity increased as fibrosis progressed from mild to severe. CONCLUSIONS: [18F]AlF-ND-bisFAPI positron emission tomography imaging is a promising noninvasive method for monitoring the progression of liver fibrosis.


Assuntos
Cirrose Hepática , Tomografia por Emissão de Pósitrons , Humanos , Ratos , Camundongos , Animais , Tomografia por Emissão de Pósitrons/métodos , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/patologia , Modelos Animais de Doenças , Biomarcadores , Fibroblastos/patologia
5.
Biotechnol J ; 19(3): e2300516, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38472100

RESUMO

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.


Assuntos
Fibroblastos , Estudo de Associação Genômica Ampla , Animais , Camundongos , Regiões Promotoras Genéticas , Genoma , Genômica , Mamíferos/genética
6.
Int J Nanomedicine ; 19: 2469-2485, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38476279

RESUMO

Background: Titanium (Ti) surface with nanotubes array via anodization has been used in dental implants to enhance bone regeneration but little research was carried out to evaluate whether the presence of highly ordered or disorderly distributed nanotubes array on titanium surface would have an effect on cell behaviors of gingival fibroblasts. Methods: The present study fabricated nanotubes arrays with varied topography under different constant voltage of electrochemical anodization in fluorine-containing electrolyte. Human gingival fibroblasts (HGFs) from extracted third molar were harvested and co-cultured with titanium disks with different nanotubes topography. Then cell behaviors of gingival fibroblasts including cell proliferation, adhesive morphology and cell migration were estimated to investigate the influence of titanium nanotubes on cell biology. Besides, gene and protein expression of adhesion molecule (integrin ß1/ß4/α6, fibronectin, intracellular adhesion molecule-1 and collagen type I) were detected to evaluate the influence of different surfaces on cell adhesion. Results: Highly ordered arrays of nanotubes with pore diameter of 60 nm and 100 nm were fabricated under 30 and 40 V of anodization (TNT-30 and TNT-40) while disorderedly distributed nanotube arrays formed on the titanium surface under 50 V of anodization (TNT-50). Our results demonstrated that compared with raw titanium surface and disorderly nanotubes, surface with orderly nanotubes array increased cell area and aspect ratio, as well as cell migration ability in the early phase of cell adhesion (p<0.05). Besides, compared with raw titanium surface, gene and protein expression of adhesion molecules were upregulated in nanotubes groups to different extents, no matter whether in an orderly or disorderly array. Conclusion: Within the limitations of our study, we conclude that compared with raw titanium surface, the presence of nanotubes array on titanium surface could enhance cells adhesion and cell migration in the early phase. And compared with disorderly distributed nanotubes, highly ordered nanotubes array might provide a much more favorable surface for gingival fibroblasts to achieve a tight adhesion on the materials.


Assuntos
Implantes Dentários , Nanotubos , Humanos , Adesão Celular , Titânio/química , Propriedades de Superfície , Fibroblastos , Nanotubos/química , Proliferação de Células
7.
Chem Biol Drug Des ; 103(3): e14454, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38477392

RESUMO

Asiatic acid (AA) is generally recognized in the treatment of various diseases and has significant advantages in the treatment of various inflammatory diseases. The treatment of rheumatoid arthritis (RA) with AA is a completely new entry point. RA is a complex autoimmune inflammatory disease, and despite the involvement of different immune and nonimmune cells in the pathogenesis of RA, fibroblast-like synoviocytes (FLS) play a crucial role in the progression of the disease. si-Nrf2 was transfected in RA-FLS and the cells were treated with AA. MTT assay and colony formation assay were used to detect the effect of AA on the viability and formation of clones of RA-FLS, respectively. Moreover, the apoptosis of RA-FLS was observed by Hoechst 33342 staining and flow cytometry. Western blot was applied to measure the expression of the Nrf2/HO-1/NF-κB signaling pathway-related proteins. Compared with the control group, RA-FLS proliferation, and clone formation were significantly inhibited by the increase of AA concentration, and further experiments showed that AA-induced apoptosis of RA-FLS. In addition, AA activated the Nrf2/HO-1 pathway to inhibit NF-κB protein expression. However, the knockdown of Nrf2 significantly offsets the effects of AA on the proliferation, apoptosis, and Nrf2/HO-1/NF-κB signaling pathway of RA-FLS cells. AA can treat RA by inhibiting the proliferation and inducing the apoptosis of RA-FLS. The mechanism may be related to the activation of the Nrf2/HO-1/NF-κB pathway.


Assuntos
Artrite Reumatoide , Triterpenos Pentacíclicos , Sinoviócitos , Humanos , NF-kappa B/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Proliferação de Células , Transdução de Sinais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Fibroblastos/metabolismo , Células Cultivadas , Apoptose
8.
J Cancer Res Clin Oncol ; 150(3): 124, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38478111

RESUMO

BACKGROUND: Cancer-associated fibroblasts (CAF) play a critical role in promoting tumor growth, metastasis, and immune evasion. While numerous studies have investigated CAF, there remains a paucity of research on their clinical application in colorectal cancer (CRC). METHODS: In this study, we collected differentially expressed genes between CAF and normal fibroblasts (NF) from previous CRC studies, and utilized machine learning analysis to differentiate two distinct subtypes of CAF in CRC. To enable practical application, a CAF-related genes (CAFGs) scoring system was developed based on multivariate Cox regression. We then conducted functional enrichment analysis, Kaplan-Meier plot, consensus molecular subtypes (CMS) classification, and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to investigate the relationship between the CAFGs scoring system and various biological mechanisms, prognostic value, tumor microenvironment, and response to immune checkpoint blockade (ICB) therapy. Moreover, single-cell transcriptomics and proteomics analyses have been employed to validate the significance of scoring system-related molecules in the identity and function of CAF. RESULTS: We unveiled significant distinctions in tumor immune status and prognosis not only between the CAF clusters, but also across high and low CAFGs groups. Specifically, patients in CAF cluster 2 or with high CAFGs scores exhibited higher CAF markers and were enriched for CAF-related biological pathways such as epithelial-mesenchymal transition (EMT) and angiogenesis. In addition, CAFGs score was identified as a risk index and correlated with poor overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS). High CAFGs scores were observed in patients with advanced stages, CMS4, as well as lymphatic invasion. Furthermore, elevated CAFG scores in patients signified a suppressive tumor microenvironment characterized by the upregulation of programmed death-ligand 1 (PD-L1), T-cell dysfunction, exclusion, and TIDE score. And high CAFGs scores can differentiate patients with lower response rates and poor prognosis under ICB therapy. Notably, single-cell transcriptomics and proteomics analyses identified several molecules related to CAF identity and function, such as FSTL1, IGFBP7, and FBN1. CONCLUSION: We constructed a robust CAFGs score system with clinical significance using multiple CRC cohorts. In addition, we identified several molecules related to CAF identity and function that could be potential intervention targets for CRC patients.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Proteínas Relacionadas à Folistatina , Humanos , Multiômica , Fibroblastos , Algoritmos , Neoplasias Colorretais/genética , Microambiente Tumoral/genética , Prognóstico
9.
Int J Biol Sci ; 20(5): 1617-1633, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481810

RESUMO

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.


Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Proliferação de Células/genética , Homeostase/genética , Fibroblastos/metabolismo , Cartilagem/metabolismo , Células Cultivadas , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo
10.
Int J Biol Sci ; 20(5): 1796-1814, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481818

RESUMO

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-ß1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-ß/Smad2/3 signaling pathway.


Assuntos
Anidrases Carbônicas , Infarto do Miocárdio , Humanos , Miocárdio/metabolismo , Proteína Smad7/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Transdução de Sinais/genética , Miócitos Cardíacos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Colágeno/metabolismo , Anidrases Carbônicas/metabolismo , Fibroblastos/metabolismo
11.
Cell Death Dis ; 15(3): 207, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472183

RESUMO

The pathogenesis of psoriasis, a chronic inflammatory autoimmune skin disease with a high global prevalence, remains unclear. We performed a high-resolution single-cell RNA sequencing analysis of 94,759 cells from 13 samples, including those from psoriasis model mice and wild-type mice. We presented a single-cell atlas of the skin of imiquimod-induced mice with psoriasis and WT mice, especially the heterogeneity of keratinocytes and fibroblasts. More interestingly, we discovered that special keratinocyte subtypes and fibroblast subtypes could interact with each other through epithelial-mesenchymal transition and validated the results with drug verification. Moreover, we conducted a tentative exploration of the potential pathways involved and revealed that the IL-17 signalling pathway may be the most relevant pathway. Collectively, we revealed the full-cycle landscape of key cells associated with psoriasis and provided a more comprehensive understanding of the pathogenesis of psoriasis.


Assuntos
Psoríase , Análise da Expressão Gênica de Célula Única , Animais , Camundongos , Queratinócitos/metabolismo , Psoríase/patologia , Pele/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C
12.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473700

RESUMO

Plant-derived nanovesicles have been considered interesting in medicine for their breakthrough biological effects, including those relevant to wound healing. However, tomato-derived nanovesicles (TDNVs) have not been studied for their effects on wound closure yet. TDNVs were isolated from Solanum lycopersicum (var. Piccadilly) ripe tomatoes by ultracentrifugation. Extract (collected during the isolation procedure) and NVs (pellet) were characterized by transmission electron microscopy and laser Doppler electrophoresis. Wound healing in the presence of Extract or NVs was analyzed by a scratch assay with monocultures of human keratinocytes (HUKE) or NIH-3T3 mouse fibroblasts. Cell proliferation and migration were studied by MTT and agarose spot assay, respectively. The vesicles in the Extract and NV samples were nanosized with a similar mean diameter of 115 nm and 130 nm, respectively. Both Extract and NVs had already accelerated wound closure of injured HUKE and NIH-3T3 monocultures by 6 h post-injury. Although neither sample exerted a cytotoxic effect on HUKE and NIH-3T3 fibroblasts, they did not augment cell proliferation. NVs and the Extract increased cell migration of both cell types. NVs from tomatoes may accelerate wound healing by increasing keratinocyte and fibroblast migration. These results indicate the potential therapeutic usefulness of TDNVs in the treatment of chronic or hard-to-heal ulcers.


Assuntos
Solanum lycopersicum , Camundongos , Animais , Humanos , Queratinócitos , Cicatrização , Fibroblastos/metabolismo , Movimento Celular , Proliferação de Células , Extratos Vegetais/metabolismo
13.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473720

RESUMO

The currently available anti-cancer therapies, such as gamma-radiation and chemotherapeutic agents, induce cell death and cellular senescence not only in cancer cells but also in the adjacent normal tissue. New anti-tumor approaches focus on limiting the side effects on normal cells. In this frame, the potential anti-tumor properties of Pulsed Electromagnetic Fields (PEMFs) through the irradiation of breast cancer epithelial cells (MCF-7 and MDA-MB-231) and normal fibroblasts (FF95) were investigated. PEMFs had a frequency of 8 Hz, full-square wave type and magnetic flux density of 0.011 T and were applied twice daily for 5 days. The data collected showcase that PEMF application decreases the proliferation rate and viability of breast cancer cells while having the opposite effect on normal fibroblasts. Moreover, PEMF irradiation induces cell death and cellular senescence only in breast cancer cells without any effect in the non-cancerous cells. These findings suggest PEMF irradiation as a novel, non-invasive anti-cancer strategy that, when combined with senolytic drugs, may eliminate both cancer and the remaining senescent cells, while simultaneously avoiding the side effects of the current treatments.


Assuntos
Neoplasias da Mama , Campos Eletromagnéticos , Humanos , Feminino , Morte Celular , Senescência Celular , Fibroblastos
14.
Int J Mol Sci ; 25(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38473788

RESUMO

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide, with high morbidity and mortality rates. The evidence for the tumor-supporting capacities of cancer-associated fibroblasts (CAFs) that modulate cancer cell proliferation, invasion, metastasis, and tumor immunity, including in CRC, has been attracting attention. The present study examined the expression status of CD70 and POSTN in CRC and analyzed their association with clinicopathological features and clinical outcomes. In the present study, in total 15% (40/269) and 44% (119/269) of cases exhibited CD70 and POSTN expression on CAFs, respectively. Co-expression of CD70 and POSTN was detected in 8% (21/269) of patients. Fluorescent immunohistochemistry identified the co-expression of CD70 and POSTN with FAP and PDPN, respectively. ACTA2 was not co-expressed with CD70 or POSTN in CRC CAFs. CRC with CD70+/POSTN+ status in CAFs was significantly associated with distant organ metastasis (p = 0.0020) or incomplete resection status (p = 0.0011). CD70+/POSTN+ status tended to associate with advanced pT stage (p = 0.032) or peritoneal metastasis (p = 0.0059). Multivariate Cox hazards regression analysis identified CD70+/POSTN+ status in CAFs [hazard ratio (HR) = 3.78] as a potential independent risk factor. In vitro experiments revealed the activated phenotypes of colonic fibroblasts induced by CD70 and POSTN, while migration and invasion assays identified enhanced migration and invasion of CRC cells co-cultured with CD70- and POSTN-expressing colonic fibroblasts. On the basis of our observations, CD70 and POSTN immunohistochemistry can be used in the prognostication of CRC patients. CRC CAFs may be a promising target in the treatment of CRC patients.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos/metabolismo , Imuno-Histoquímica , Proliferação de Células , Neoplasias Colorretais/patologia , Moléculas de Adesão Celular/metabolismo , Ligante CD27/metabolismo
15.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474040

RESUMO

Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease's pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.


Assuntos
Escleroderma Sistêmico , Doenças Vasculares , Animais , Humanos , Células Endoteliais/patologia , Pele/patologia , Escleroderma Sistêmico/patologia , Fibrose , Doenças Vasculares/patologia , Fibroblastos/patologia , Células Cultivadas
16.
Int J Mol Sci ; 25(5)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38474135

RESUMO

Nucleotides, glycosaminoglycans, and omega-3 essential fatty acids (O3s) could be used for improving skin health, although their modes of action, alone or in combination, are not yet fully understood. To gain some insight into these mechanisms, we performed two in vitro tests and one in vivo pilot trial. The effects on human dermal fibroblast proliferation and migration were evaluated with the following compounds and combinations: 0.156 mg/mL O3s, 0.0017 mg/mL hyaluronic acid (HA), 0.0004 mg/mL dermatan sulfate (DS), 0.0818 mg/mL nucleotides, and [O3s + HA + DS] and [O3s + HA + DS + nucleotides] at the same concentrations. In both in vitro assays, adding nucleotides to [O3s + HA + DS] provided significant improvements. The resulting combination [O3s + HA + DS + nucleotides] was then tested in vivo in dogs with atopic dermatitis by oral administration of a supplement providing a daily amount of 40 mg/kg nucleotides, 0.9 mg/kg HA, 0.18 mg/kg DS, 53.4 mg/kg EPA, and 7.6 mg/kg DHA. After 30 days, the pruritus visual analog scale (pVAS) score was significantly reduced, and no adverse effects were observed. In conclusion, the combination of nucleotides plus glycosaminoglycans and O3s could serve as a useful therapeutic alternative in skin health applications.


Assuntos
Dermatite Atópica , Doenças do Cão , Ácidos Graxos Ômega-3 , Humanos , Animais , Cães , Dermatite Atópica/tratamento farmacológico , Saccharomyces cerevisiae , Doenças do Cão/tratamento farmacológico , Prurido/tratamento farmacológico , Ácidos Graxos Ômega-3/uso terapêutico , Glicosaminoglicanos/uso terapêutico , Ácido Hialurônico/uso terapêutico , Proliferação de Células , Fibroblastos
17.
Cells ; 13(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38474342

RESUMO

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.


Assuntos
Colágeno Tipo VI , Mecanotransdução Celular , Distrofias Musculares , Esclerose , Humanos , Colágeno Tipo VI/genética , Proteínas Hedgehog/metabolismo , Tendões/metabolismo , Fibroblastos/metabolismo
18.
Molecules ; 29(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38474467

RESUMO

Isoflavones, belonging to polyphenolic compounds, show structural similarity to natural estrogens, and in this context, they have been extensively studied. Some of them are also applied as cosmetic additives; however, little is known regarding their effects on skin cells. In this investigation, common isoflavones, including genistein, daidzein, glycitein, formononetin, and biochanin A, as well as coumestrol, were evaluated for antioxidant activity and their impact on human skin fibroblasts and keratinocytes. Antioxidant effects were assessed using DPPH, ABTS, and FRAP tests, and the ability to scavenge reactive oxygen species (ROS) was tested in cells with H2O2-provoked oxidative stress. The impact on the activity of antioxidant enzymes (SOD, CAT, GSH) and lipid peroxidation (MDA) was also explored. As shown by Alamar Blue and neutral red uptake assays, the compounds were not toxic within the tested concentration range, and formononetin and coumestrol even demonstrated a stimulatory effect on cells. Coumestrol and biochanin A demonstrated significant antioxidative potential, leading to a significant decrease in ROS in the cells stimulated by H2O2. Furthermore, they influenced enzyme activity, preventing depletion during induced oxidative stress, and also reduced MDA levels, demonstrating protection against lipid peroxidation. In turn, genistein, daidzein, and glycitein exhibited low antioxidant capacity.


Assuntos
Genisteína , Isoflavonas , Humanos , Genisteína/farmacologia , Cumestrol , Espécies Reativas de Oxigênio , Fitoestrógenos , Antioxidantes , Peróxido de Hidrogênio , Isoflavonas/química , Estresse Oxidativo , Queratinócitos , Fibroblastos
19.
Exp Dermatol ; 33(3): e15040, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38429888

RESUMO

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.


Assuntos
Amiloidose , Epidermólise Bolhosa Distrófica , Interleucina-6 , Humanos , Amiloidose/metabolismo , Amiloidose/patologia , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Fibroblastos/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Proteína Amiloide A Sérica/metabolismo , Receptores Toll-Like/metabolismo
20.
J Vis Exp ; (204)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38465922

RESUMO

Mutations in the autophagy gene WDR45/WIPI4 are the cause of beta-propeller-associated neurodegeneration (BPAN), a subtype of human diseases known as neurodegeneration with brain iron accumulation (NBIA) due to the presence of iron deposits in the brains of patients. Intracellular iron levels are tightly regulated by a number of cellular mechanisms, including the critical mechanism of ferritinophagy. This paper describes how ferritinophagy can be assessed in primary, skin-derived human fibroblasts. In this protocol, we use iron-modulating conditions for inducing or inhibiting ferritinophagy at the cellular level, such as the administration of bafilomycin A1 to inhibit lysosome function and ferric ammonium citrate (FAC) or deferasiox (DFX) treatments to overload or deplete iron, respectively. Such treated fibroblasts are then subjected to high-throughput imaging and CellProfiler-based quantitative localization analysis of endogenous ferritin and autophagosomal/lysosomal markers, here LAMP2. Based on the level of autophagosomal/lysosomal ferritin, conclusions can be drawn regarding the level of ferritinophagy. This protocol can be used to assess ferritinophagy in BPAN patient-derived primary fibroblasts or other types of mammalian cells.


Assuntos
Ferritinas , Ferro , Animais , Humanos , Ferro/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Encéfalo/metabolismo , Autofagia , Fibroblastos/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte/metabolismo
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