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1.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1350306

RESUMO

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Assuntos
Humanos , Corpo Vítreo , Proliferação de Células , Desdiferenciação Celular , Tretinoína , Células Tumorais Cultivadas , Diferenciação Celular , Divisão Celular , Linhagem Celular
2.
Theranostics ; 12(12): 5470-5487, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35910794

RESUMO

Rationale: Wound healing is among the most complicated physiological processes and requires the synchronization of various cell types with distinct roles to re-establish the condition of the original skin. Patients affected by peripheral neuropathies often experience failure to heal. Loss of Schwann cells (SCs), a crucial population of peripheral nervous system cells in skin, may contribute to chronic wounds. However, the role of SCs in wound healing are poorly understood. Methods: The activity of SCs was investigated by using a cell atlas of the wound healing process, which was generated by integrating single-cell RNA sequencing (scRNA-seq) libraries covering different states of mouse back skin. The results of in silico analysis were validated by in vitro cell culture and in vivo mouse model. Selective inhibitors and conditional RNAi by virus transfection were utilized to investigate the role of SCs in wound healing. Findings from mouse experiments were further verified in scRNA-seq analysis of diabetic patients. Results: Our in silico analysis revealed the heterogeneous cellular components of skin and the dynamic interactions of neural crest derived cells (NCs) with other cell types. We found that SCs dedifferentiated at an early stage of wound repair with upregulated Wnt signaling. We also identified dedifferentiated SC (dSC) defect in diabetic wounds in both mouse and human. Wnt inhibition at the wound site repressed SC dedifferentiation, leading to defective repair. Furthermore, dSCs derived TGF-ß3, which is context-dependent, promoted the migration of fibroblasts and keratinocytes. Moreover, TGF-ß3 supplementation enhanced the healing of chronic wounds in diabetic mice with impaired SCs. Conclusion: Our study thus advances the understanding of the roles of neural-derived cells in skin regeneration and suggests a potential therapeutic strategy for wound healing disorders.


Assuntos
Desdiferenciação Celular , Diabetes Mellitus Experimental , Doenças do Sistema Nervoso Periférico , Células de Schwann , Fator de Crescimento Transformador beta3 , Cicatrização , Animais , Desdiferenciação Celular/genética , Desdiferenciação Celular/fisiologia , Humanos , Camundongos , Doenças do Sistema Nervoso Periférico/genética , Células de Schwann/fisiologia , Pele/lesões , Pele/inervação , Fator de Crescimento Transformador beta3/genética , Cicatrização/genética , Cicatrização/fisiologia
3.
J Agric Food Chem ; 70(24): 7420-7440, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35687823

RESUMO

The activation of platelets and proliferation of vascular smooth muscle cells (VSMCs) in the vascular intima play an essential role in the pathological mechanism of vascular restenosis (RS). Rosmarinic acid (RA) is a natural phenolic acid compound. However, its mechanism of action on platelets and VSMCs is still unclear. This study investigated the effects of RA on platelet function, VSMCs phenotypic conversion, proliferation, and migration in vascular remodeling with a specific focus on the Keap1-Nrf2-ARE signaling pathway. RA inhibited platelet aggregation and Ca2+ release and significantly reduced the release of platelet microvesicles. In addition, RA inhibited the phenotypic transition of VSMCs in vitro and in vivo. In vitro experiments showed that RA could effectively inhibit the proliferation and migration of VSMCs induced by the platelet-derived growth factor (PDGF)-BB. PDGF-BB triggered ROS generation and a decrease in mitochondrial membrane potential, which were inhibited by RA. Mechanistically, after artery injury or treatment with PDGF-BB, VSMCs presented with inhibition of the Nrf2/antioxidant response element (ARE) signaling pathway. RA treatment reversed this profile by activating the Nrf2/ARE signaling pathway; stabilizing Keap1 protein; upregulating HO-1, NQO1, GCLM, and GST protein levels; promoting typical Nrf2 nuclear translocation; and preventing VSMCs from oxidative stress damage. On the other hand, RA also inhibited the NF-κB pathway to reduce inflammation. In summary, these results indicate that RA inhibits platelet function and attenuates the proliferation, migration, and phenotypic transition of VSMCs induced by PDGF-BB in vitro and vascular remodeling in vivo. Therefore, RA treatment may be a potential therapy for preventing or treating RS.


Assuntos
Músculo Liso Vascular , Fator 2 Relacionado a NF-E2 , Elementos de Resposta Antioxidante , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Becaplermina/farmacologia , Desdiferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Cinamatos , Depsídeos , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Miócitos de Músculo Liso , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neointima/metabolismo , Neointima/patologia , Neointima/prevenção & controle , Agregação Plaquetária , Remodelação Vascular
4.
Cell Rep ; 39(4): 110730, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35476977

RESUMO

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.


Assuntos
Reprogramação Celular , Fígado , Animais , Desdiferenciação Celular , Hepatócitos/metabolismo , Fígado/metabolismo , Regeneração Hepática , Mamíferos , Camundongos
5.
Mol Metab ; 60: 101493, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35398277

RESUMO

OBJECTIVE: ß cell dedifferentiation may underlie the reversible reduction in pancreatic ß cell mass and function in type 2 diabetes (T2D). We previously reported that ß cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic ß cell dedifferentiation in Sirt3-deficient ß cells. METHODS: We applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on ß cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the ß cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. RESULTS: In Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by ß cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced ß cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. CONCLUSIONS: Sirt3 prevents ß cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse ß cell dysfunction associated with glucolipotoxicity.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Sirtuína 3/metabolismo , Animais , Desdiferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Sirtuína 3/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Exp Cell Res ; 415(1): 113109, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35318048

RESUMO

Tissue engineering technology will be the main approach to tissue regeneration in the future and are promising for the treatment of large-area burns and refractory wounds. Dedifferentiated fat cells (DFAT) derived from mature adipocytes (MAs) as a seed cell have great potential in cell therapy and tissue engineering for the treatment of a variety of clinical diseases because of their wider availability, stronger proliferation ability, multidirectional redifferentiation potential, higher cell purity, lower heterogeneity, and greater biosafety profile. However, the triggering mechanism for MAs reprogramming in vitro is unclear. In this study, MAs were successfully induced to dedifferentiate into DFAT in a short time in vitro using an "improved ceiling culture method". Flow cytometry, adipogenic, and osteogenic differentiation experiments verified that DFAT cells present the biological characteristics of stem cells. In addition, changing the stiffness of the extracellular matrix can inhibit the dedifferentiation of MAs to DFAT, and increase the expression of Yes-associated protein/transcriptional co-activator with the PDZ-binding motif (YAP/TAZ), nuclear translocation, and the expression of reprogramming transcription factors. In conclusion, extracellular matrix stiffness can induce MAs to dedifferentiate into DFAT in vitro, and can directly transmit mechanical force signals to the nucleus via YAP/TAZ binding to trigger the expression of stem cell-related reprogramming factors.


Assuntos
Desdiferenciação Celular , Osteogênese , Adipócitos/metabolismo , Adipogenia , Diferenciação Celular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
J Genet Genomics ; 49(4): 316-328, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35292418

RESUMO

Type 2 diabetes (T2D) is caused by insulin resistance and insufficient insulin secretion. Evidence has increasingly indicated that pancreatic ß-cell dysfunction is the primary determinant of T2D disease progression and remission. High plasticity is an important feature of pancreatic ß-cells. During T2D development, pancreatic ß-cells undergo dynamic adaptation. Although ß-cell death/apoptosis in later-stage T2D is the major cause of ß-cell dysfunction, recent studies have revealed that ß-cell dedifferentiation and reprogramming, which play critical roles in ß-cell functional regulation in the early and middle T2D progression stages, are characterized by (i) a loss of mature ß-cell-enriched genes; (ii) dedifferentiation to a progenitor-like state; and (iii) transdifferentiation into other cell types. The roles of transcription factors (TFs) in the establishment and maintenance of ß-cell identity during pancreatic development have been extensively studied. Here, we summarize the roles and underlying mechanisms of TFs in the maintenance of ß-cell identity under physiological and type 2 diabetic conditions. Several feasible approaches for restoring islet functions are also discussed. A better understanding of the transcriptional control of ß-cell identity and plasticity will pave the way for developing more effective strategies, such as ß-cell regeneration therapy, to treat T2D and associated metabolic disorders.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Desdiferenciação Celular/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo
8.
Indian J Pathol Microbiol ; 65(1): 65-75, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35074968

RESUMO

BACKGROUND: Liposarcomas including atypical lipomatous tumors (ALT)/well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPSs) display a histomorphological spectrum with their several diagnostic mimics. Murine double minute 2(MDM2) gene amplification characterizes ALT/WDLPS and DDLPS. Presently, there is no documented study from our subcontinent on the validation of MDM2 gene testing in these tumors. MATERIAL AND METHODS: Twenty-eight cases, diagnosed as ALT/WDLPS (n = 5) and DDLPSs (n = 23), along with 10 other tumors were tested for MDM2 gene amplification, using fluorescence in situ hybridization (FISH) on tissue microarrays (TMAs). Fourteen cases, diagnosed as ALT/WDLPS and DDLPS, along with 49 other tumors were tested for MDM2 immunostaining. Twenty tumors were tested for p16INK4a immunostaining. RESULTS: FISH was interpretable in 25 (89.2%) cases. Among the 20 cases diagnosed as DDLPSs, 19 displayed MDM2 gene amplification. Among the 5 cases diagnosed as ALT/WDLPS, four showed MDM2 gene amplification. Finally, 19 cases were confirmed as DDLPS and 4 as ALT/WDLPS. Furthermore, 7/19 cases confirmed as DDLPS and all 4 cases as ALT/WDLPS tested for MDM2 immunostaining, displayed its diffuse immunoexpression, while a single case of DDLPS showed its focal immunostaining. None of the 49 control cases displayed diffuse MDM2 immunoexpression. ALL 16 DDLPSs and 4 cases of ALT/WDLPS displayed p16INK4a immunostaining. The sensitivity for diffuse MDM2 immunostaining was 87.5% in cases of DDLPS, 100% in ALT/WDLPS, and specificity was 100%. The sensitivity for MDM2 gene amplification was 94.7% in cases of DDLPS and 100% in cases of ALT/WDLPS. The sensitivity for p16INK4a was 100%. CONCLUSION: This constitutes the first sizable study on MDM2 testing in ALT/WDLPS and DDLPS from our subcontinent using TMAs. MDM2 gene amplification testing continues as the diagnostic gold standard for ALTs/WDLPSs and DDLPSs and is useful in cases of diagnostic dilemmas. Diffuse MDM2 (IF2 clone) and p16INK4a immunostaining, together seem useful for triaging cases for FISH.


Assuntos
Hibridização in Situ Fluorescente/métodos , Lipossarcoma/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Proto-Oncogênicas c-mdm2/genética , Análise Serial de Tecidos/métodos , Adulto , Idoso , Biomarcadores Tumorais/genética , Desdiferenciação Celular , Feminino , Humanos , Lipossarcoma/classificação , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/normas , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Análise Serial de Tecidos/normas
9.
Immunogenetics ; 74(2): 231-244, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35001141

RESUMO

Effector T cells, which are abundant but are short-lived after reinfusion into the body, are generally used for T-cell therapy, and antitumor immunity is typically not maintained over the long term. Genetic modification by early differentiated T cells and reinfusion has been shown to enhance antitumor immunity in vivo. This study overexpressed the characteristic transcription factors of differentiated early T cells by transfecting effector T cells with transcription factor recombinant lentivirus (S6 group: BCL6, EOMES, FOXP1, LEF1, TCF7, KLF7; S1 group: BCL6, EOMES, FOXP1, KLF7; S3 group: BCL6, EOMES, FOXP1, LEF1) to induce a sufficient number of effector T cells to dedifferentiate and optimize the transcription factor system. The results revealed that overexpression of early characteristic transcription factors in effector T cells upregulated the expression of early T cell differentiation markers (CCR7 and CD62L), with the S1 group having the highest expression level, while the rising trend of late differentiation marker (CD45RO) expression was suppressed. Moreover, the expression of early differentiation-related genes (ACTN1, CERS6, BCL2) was significantly increased, while the expression of late differentiation-related genes (KLRG-1) and effector function-related genes (GNLY, GZMB, PRF1) was significantly decreased; this difference in expression was more significant in the S1 group than in the other two experimental groups. The antiapoptotic ability of each experimental group was significantly enhanced, while the secretion ability of TNF-α and IFN-γ was weakened, with the effector cytokine secretion ability of the S1 group being the weakest. Transcriptomic analysis showed that the gene expression profile of each experimental group was significantly different from that of the control group, with differences in the gene expression pattern and number of differentially expressed genes in the S1 group compared with the other two experimental groups. The differentially expressed gene enrichment pathways were basically related to the cell cycle, cell division, and immune function. In conclusion, overexpression of early characteristic transcription factors in effector T cells induces their dedifferentiation, and induction of dedifferentiation by the S1 group may be more effective.


Assuntos
Desdiferenciação Celular , Fatores de Transcrição , Linfócitos T CD8-Positivos , Desdiferenciação Celular/genética , Diferenciação Celular/genética , Fenótipo , Subpopulações de Linfócitos T , Fatores de Transcrição/genética
10.
Obesity (Silver Spring) ; 30(2): 447-459, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35088561

RESUMO

OBJECTIVE: The aim of this study was to research the role and underlying mechanism of miR-195 involved in pancreatic ß-cell dedifferentiation induced by hyperlipemia in type 2 diabetes mellitus. METHODS: High-fat-diet-induced obese C57BL/6J mice and palmitate-stimulated Min6 cells were used as the models of ß-cell dedifferentiation in vivo and in vitro, respectively. The expression of miR-195 and insulin secretion during ß-cell dedifferentiation were measured. Also, the influence of regulated miR-195 expression on ß-cell dedifferentiation was examined. Meanwhile, the IRS-1/2/Pi3k/Akt pathway and mitofusin-2 (Mfn2) expression were investigated during ß-cell dedifferentiation. RESULTS: MiR-195 was upregulated during lipotoxicity-induced ß-cell dedifferentiation in both in vivo and in vitro experiments, and miR-195 functionally contributed to lipotoxicity-induced ß-cell dedifferentiation. Furthermore, miR-195 inhibited IRS-1/2/Pi3k/Akt pathway activation, which accompanied ß-cell dedifferentiation. Mfn2, a target of miR-195, was found to be downregulated and was associated with increased mitochondrial production of reactive oxygen species during ß-cell dedifferentiation. Instructively, inhibition of miR-195, at least partially, reversed the downregulation of Mfn2, restored IRS-1/2/Pi3k/Akt pathway activation, and prevented ß-cell dedifferentiation. CONCLUSIONS: MiR-195 promoted ß-cell dedifferentiation through negatively regulating Mfn2 expression and inhibiting the IRS-1/2/Pi3k/Akt pathway, providing a promising treatment for type 2 diabetes mellitus.


Assuntos
Diabetes Mellitus Tipo 2 , MicroRNAs , Animais , Desdiferenciação Celular , Diabetes Mellitus Tipo 2/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
Aesthet Surg J ; 42(6): NP423-NP431, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35032169

RESUMO

BACKGROUND: Fat transplantation is a common method employed to treat soft-tissue defects. The dedifferentiation of mature adipocytes has been well documented, but whether it occurs after fat transplantation remains unclear. OBJECTIVES: The major purpose of this project was to investigate the dedifferentiation of mature adipocytes after fat transplantation. METHODS: Human lipoaspirate tissue was obtained from 6 female patients who underwent esthetic liposuction. Mature adipocytes were extracted and labeled with PKH26, mixed with lipoaspirate, and injected into nude mice. In addition, PKH26+ adipocytes were subjected to a ceiling culture. Grafted fat was harvested from nude mice, and stromal vascular fragment cells were isolated. The immunophenotype of PKH26+ cells was detected by flow cytometry analysis at 2 days and 1 week. The PKH26+ cells were sorted and counted at 2 and 4 weeks to verify their proliferation and multilineage differentiation abilities. RESULTS: Two days after transplantation, almost no PKH26+ cells were found in the stromal vascular fragment cells. The PKH26+ cells found 1 week after transplantation showed a positive expression of cluster of differentiation (CD) 90 (CD90) and CD105 and a negative expression of CD45. This indicates that the labeled adipocytes were dedifferentiated. Its pluripotency was further demonstrated by fluorescent cell sorting and differentiation culture in vitro. In addition, the number of live PKH26+ cells at week 4 [(6.83 ± 1.67) × 104] was similar with that at week 2 [(7.11 ± 1.82) × 104]. CONCLUSIONS: Human mature adipocytes can dedifferentiate into stem cell-like cells in vivo after fat transplantation.


Assuntos
Desdiferenciação Celular , Lipectomia , Adipócitos , Tecido Adiposo/transplante , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Nus , Células-Tronco
12.
BMC Plant Biol ; 22(1): 9, 2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-34979922

RESUMO

BACKGROUND: Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated. RESULTS: In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed. CONCLUSION: This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.


Assuntos
Arabidopsis/fisiologia , Desdiferenciação Celular/genética , Parede Celular/fisiologia , MicroRNAs/genética , Células Vegetais/fisiologia , RNA de Plantas/genética , Arabidopsis/genética , MicroRNAs/metabolismo , RNA de Plantas/metabolismo
13.
PLoS One ; 17(1): e0262272, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34986184

RESUMO

High-level amplification of MDM2 and other genes in the 12q13-15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.


Assuntos
Desdiferenciação Celular/genética , Ácidos Nucleicos Livres/genética , Amplificação de Genes/genética , Lipossarcoma/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Idoso , Diferenciação Celular/genética , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias de Tecidos Moles/genética , Sequenciamento Completo do Genoma/métodos
14.
J Virol ; 96(5): e0197421, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35019721

RESUMO

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single-cell RNA sequencing (scRNA-seq) studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We show here that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a dedifferentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1-infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell redifferentiation destabilized latent infection. Guided by these findings, data mining of single-cell RNA-seq data of latently HIV-1-infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. More than 20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g., HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, and CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1-infected cells differs from that of normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.


Assuntos
Linfócitos T CD4-Positivos , Desdiferenciação Celular , Infecções por HIV , HIV-1 , Latência Viral , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos
15.
Dev Cell ; 57(2): 166-179.e6, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35016013

RESUMO

Loss of differentiated cells to tissue damage is a hallmark of many diseases. In slow-turnover tissues, long-lived differentiated cells can re-enter the cell cycle or transdifferentiate to another cell type to promote repair. Here, we show that in a high-turnover tissue, severe damage to the differentiated compartment induces progenitors to transiently acquire a unique transcriptional and morphological postmitotic state. We highlight this in an acute villus injury model in the mouse intestine, where we identified a population of progenitor-derived cells that covered injured villi. These atrophy-induced villus epithelial cells (aVECs) were enriched for fetal markers but were differentiated and lineage committed. We further established a role for aVECs in maintaining barrier integrity through the activation of yes-associated protein (YAP). Notably, loss of YAP activity led to impaired villus regeneration. Thus, we define a key repair mechanism involving the activation of a fetal-like program during injury-induced differentiation, a process we term "adaptive differentiation."


Assuntos
Adaptação Biológica/fisiologia , Desdiferenciação Celular/fisiologia , Cicatrização/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Desdiferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Feminino , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fosfoproteínas/metabolismo , Regeneração , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Proteínas de Sinalização YAP/metabolismo
16.
Am J Respir Cell Mol Biol ; 66(1): 23-37, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34236953

RESUMO

The U.S. Food and Drug Administration-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential antifibrotic actions. However, neither its in vivo efficacy in lung fibrosis nor its dependence on proteasome inhibition has been conclusively defined. In this study, we assessed the therapeutic efficacy of BTZ in a mouse model of pulmonary fibrosis, developed an in vitro protocol to define its actions on diverse fibroblast activation parameters, determined its reliance on proteasome inhibition for these actions in vivo and in vitro, and explored alternative mechanisms of action. The therapeutic administration of BTZ diminished the severity of pulmonary fibrosis without reducing proteasome activity in the lung. In experiments designed to mimic this lack of proteasome inhibition in vitro, BTZ reduced fibroblast proliferation, differentiation into myofibroblasts, and collagen synthesis. It promoted dedifferentiation of myofibroblasts and overcame their characteristic resistance to apoptosis. Mechanistically, BTZ inhibited kinases important for fibroblast activation while inducing the expression of DUSP1 (dual-specificity protein phosphatase 1), and knockdown of DUSP1 abolished its antifibrotic actions in fibroblasts. Collectively, these findings suggest that BTZ exhibits a multidimensional profile of robust inhibitory actions on lung fibroblasts as well as antifibrotic actions in vivo. Unexpectedly, these actions appear to be independent of proteasome inhibition, instead attributable to the induction of DUSP1.


Assuntos
Bortezomib/uso terapêutico , Fibroblastos/patologia , Inibidores de Proteassoma/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Adulto , Apoptose/efeitos dos fármacos , Bleomicina , Bortezomib/farmacologia , Desdiferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , NF-kappa B/metabolismo , Prostaglandinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Receptor fas/metabolismo
17.
J Genet Genomics ; 49(3): 208-216, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34861413

RESUMO

Decreased functional ß-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. ß-cell-specific knockout of Grb10 in mice increased ß-cell mass and improved ß-cell function. Grb10-deficient ß-cells exhibit enhanced mTORC1 signaling and reduced ß-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced ß-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of ß-cell dedifferentiation and ß-cell mass, which exerts its effect by inhibiting mTORC1 signaling.


Assuntos
Diabetes Mellitus Experimental , Proteína Adaptadora GRB10 , Animais , Desdiferenciação Celular/genética , Proliferação de Células/genética , Proteína Adaptadora GRB10/genética , Proteína Adaptadora GRB10/metabolismo , Insulina/metabolismo , Camundongos
18.
Pigment Cell Melanoma Res ; 35(1): 18-25, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34407291

RESUMO

Melanoma is the deadliest form of skin cancer. Although treatment with targeted therapies and immune checkpoint inhibitors has dramatically improved survival in advanced melanoma, many patients do not benefit from these therapies or relapse after an initial period of response. Thus, future outcomes in these categories of melanoma patients will depend on the identification of novel therapeutic targets and methods to enhance existing targeted therapy and immunotherapy regimens. Ferroptosis is a newly identified form of iron-dependent regulated cell death that is morphologically, biochemically, and genetically distinct from apoptosis, autophagy, pyroptosis, and necroptosis. Dysregulation of ferroptosis has been linked to the development of several forms of cancer. This review examines ferroptosis in the context of melanoma. It presents an overview of ferroptosis biology, summarizes and interprets the current literature, and poses several outstanding questions and areas of future direction.


Assuntos
Ferroptose , Melanoma/patologia , Neoplasias Cutâneas/patologia , Animais , Desdiferenciação Celular , Resistencia a Medicamentos Antineoplásicos , Ferroptose/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Ferro/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo
19.
Pigment Cell Melanoma Res ; 35(1): 38-51, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34467641

RESUMO

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.


Assuntos
Desdiferenciação Celular , Plasticidade Celular , Melanócitos/fisiologia , Pele/citologia , Adulto , Proliferação de Células , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Melaninas/metabolismo , Melanócitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Nestina/genética , Nestina/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA-Seq , Transdução de Sinais , Transcriptoma , Adulto Jovem
20.
Ann Vasc Surg ; 79: 335-347, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34648856

RESUMO

BACKGROUND: Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS: Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS: An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION: SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.


Assuntos
Desdiferenciação Celular , Citoesqueleto/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Tromboflebite/patologia , Varizes/patologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Estudos de Casos e Controles , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Senescência Celular , Citoesqueleto/metabolismo , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Veia Safena/metabolismo , Veia Safena/patologia , Tromboflebite/genética , Tromboflebite/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Varizes/genética , Varizes/metabolismo
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