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Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.
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Electricidad , Fibroblastos , Piel , Actinas/biosíntesis , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Terapia por Estimulación Eléctrica , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/fisiología , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Piel/citologíaRESUMEN
Endogenous electric field (EF)-directed keratinocytes migration is known to play a key role in the wound re-epithelialization process. Although many molecules and signaling pathways are reported important for directional keratinocytes migration under EF, the underlying mechanism remains unclear. Our previous research found that CD9, a trans-membrane protein, is involved in wound re-epithelialization and CD9 downregulation contributes to keratinocytes migration. In this study, we observed the effect of EF on CD9 expression and keratinocytes migration. The keratinocytes migrated directionally toward the cathode and CD9 expression was down-regulated under EF (200mV/mm). In addition, CD9 overexpression reversed EF-induced migratory speed and the electrotactic response of keratinocytes. Also, we found that EF reduced AMP-activated protein kinase (AMPK) activity. Furthermore, AICAR, an AMPK activator, increased CD9 expression under EF, while compound C, an AMPK inhibitor, decreased CD9 expression in keratinocytes. Our results demonstrate that EF regulates CD9 expression and keratinocytes directional migration, in which AMPK pathway plays an important role.
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Proteínas Quinasas Activadas por AMP/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Tetraspanina 29/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Regulación hacia Abajo , Estimulación Eléctrica/métodos , Humanos , Queratinocitos/química , Redes y Vías Metabólicas , Ratones Endogámicos BALB C , Tetraspanina 29/genéticaRESUMEN
Immune checkpoint inhibitors have revolutionized the treatments of lung cancers, and multiple predictive biomarkers alone or in combination help clinicians with the appropriate therapeutic selections. Recently, chemo-immunotherapy has been recommended for treating advanced non-small cell lung cancers in patients without driver mutations. However, the clinical relevance of predictive biomarkers and the treatment efficacy of chemo-immunotherapy in large cell lung carcinoma (LCLC) remain unclear. Here, we reported a rare case of LCLC with none driver gene mutations and low values of multiple predictive biomarkers. These biomarkers included a low PD-L1 expression of 5-10%, a low tumor mutational burden (TMB) of 2.5 muts/mb, a low CD8(+) tumor-infiltrating lymphocyte density of 147.91 psc/mm². After one-cycle chemotherapy, the patient progressed rapidly and then was switched to pembrolizumab combining paclitaxel plus cisplatin. Interestingly, he achieved a partial response after two cycles of chemo-immunotherapy, showing multiple lymph nodes obviously shrunk on CT scan, and other clinical symptoms were relieved when compared with the baseline findings. After five cycles of chemo-immunotherapy, this advanced patient still benefited and was changed to maintenance immunotherapy monotherapy. This case suggests that chemo-immunotherapy may provide an effective therapeutic option for those LCLC patients with low values of multiple predictive biomarkers, particularly for those who progressed from first-line classical treatments.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Carcinoma de Células Grandes/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Adulto , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Células Grandes/inmunología , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/patología , Cisplatino/uso terapéutico , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Paclitaxel/uso terapéutico , Inducción de Remisión , Resultado del Tratamiento , Microambiente TumoralRESUMEN
During keratinocyte stratification and wound healing, keratinocytes undergo a switch between differentiation and motility. However, limited knowledge exists on the mechanisms of the switch. We have previously demonstrated that the expression of CD9 was changed in different wound stages and involved in the regulation of keratinocyte migration. In this study, we showed that CD9 expression was increased in both human and mouse keratinocytes undergoing differentiation. CD9 overexpression in keratinocytes stimulated terminal differentiation and reduced cell motility. CD9 silencing inhibited calcium-induced keratinocyte differentiation and increased cell motility. Furthermore, CD9 overexpression recruited E-cadherin to the plasma membrane and subsequently activated PI3K/Akt signaling, while CD9 knockdown inhibited the recruitment of E-cadherin to the plasma membrane and PI3K/Akt activation. Importantly, silencing E-cadherin expression or inhibiting PI3K/Akt signaling reversed CD9 overexpression-induced differentiation and -reduced motility. These results demonstrate that CD9 acts as an important node that regulates keratinocyte differentiation and motility. The recruitment of E-cadherin to the plasma membrane and activation of the PI3K/Akt signaling pathway mediated by CD9 play an important role in these processes.
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Cadherinas/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Transducción de Señal , Tetraspanina 29/metabolismo , Animales , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Humanos , Queratina-10/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Piel/patología , Tetraspanina 29/antagonistas & inhibidores , Tetraspanina 29/genéticaRESUMEN
With the development and application of next-generation sequencing (NGS) and target capture technology, the demand for an effective analysis method to accurately detect gene fusion from high-throughput data is growing. Hence, we developed a novel fusion gene analyzing method called single-end gene fusion (SEGF) by starting with single-end DNA-seq data. This approach takes raw sequencing data as input, and integrates the commonly used alignment approach basic local alignment search tool (BLAST) and short oligonucleotide analysis package (SOAP) with stringent passing filters to achieve successful fusion gene detection. To evaluate SEGF, we compared it with four other fusion gene discovery analysis methods by analyzing sequencing results of 23 standard DNA samples and DNA extracted from 286 lung cancer formalin fixed paraffin embedded (FFPE) samples. The results generated by SEGF indicated that it not only detected the fusion genes from standard samples and clinical samples, but also had the highest accuracy and sensitivity among the five compared methods. In addition, SEGF was capable of detecting complex gene fusion types from single-end NGS sequencing data compared with other methods. By using SEGF to acquire gene fusion information at DNA level, more useful information can be retrieved from the DNA panel or other DNA sequencing methods without generating RNA sequencing information to benefit clinical diagnosis or medication instruction. It was a timely and cost-effective measure with regard to research or diagnosis. Considering all the above, SEGF is a straightforward method without manipulating complicated arguments, providing a useful approach for the precise detection of gene fusion variation.
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In this study, we applied various developmental stages of zebrafish to address the potential environmental risk and aquatic toxicity of bromothalonil and flutolanil. This results demonstrated that the acute toxicity of bromothalonil to the three phases of zebrafish were 4.34 (embryo) < 3.27 (12 h old larvae) < 2.52 mg/L (adult fish) and that of flutolanil were 5.47 (embryo) < 4.09 (72 h old larvae) < 3.91 (12 h old larvae) < 2.70 mg/L (adult). Sublethal effects induced by both bromothalonil and flutolanil on zebrafish embryos were noted, including growth inhibition, abnormal spontaneous movement, slower heart rate, complete hatching failure, and morphological deformities. In addition, both bromothalonil and flutolanil could cause notochord deformation and short body length of larvae. This study provides a foundation for future investigation into the mechanism of bromothalonil and flutolanil toxicity in zebrafish.
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Anilidas/toxicidad , Fungicidas Industriales/toxicidad , Nitrilos/toxicidad , Pez Cebra/fisiología , Animales , Embrión no Mamífero/efectos de los fármacos , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/fisiología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrolloRESUMEN
Primary studies in animal models and humans have suggested the therapeutic potential of autologous stem cell for treating chronic lower extremity ulcers. However, the results of pilot randomized controlled trials (RCTs) in humans have been inconsistent. A meta-analysis of RCTs was performed to evaluate the role of autologous stem cell-based therapy for lower extremity ulcers.Studies were identified during a systematic search of Medline, Embase, Cochrane's library, and references cited in related reviews and studies. Studies were included if they were RCTs published in English, recruited patients with lower extremity ulcers who were assigned to either a group for the topical therapy with autologous stem cells, and reported data regarding the healing of the ulcers.Relative risks (RRs) for healing rate and standardized mean differences (SMDs) for the changes in the mean sizes of ulcers were evaluated with a random-effects model. Overall, autologous stem cell-based therapy was associated with better healing of lower extremity ulcers (12 comparisons, 290 patients, RR for partial healingâ=â3.07, 95% confidence interval [CI]â=â1.14-8.24, Pâ=â0.03; RR for complete healingâ=â2.26, 95% CIâ=â1.48-3.16, Pâ<â0.001) with little heterogeneity (Iâ=â0%). Moreover, autologous stem cell-based therapy was associated with a greater reduction in mean ulcer size (SMDâ=â-0.63, 95% CIâ=â-1.03 to -0.22, Pâ=â0.002). Subgroup analyses indicated that stem cells from peripheral blood and bone marrow seemed to exert similar beneficial effects on the healing of ulcers. Stem cell therapy was not associated with any increased risks for adverse events. The optimized sources, amounts, and delivery methods of stem cell -based therapy for patients with chronic lower extremity ulcers need to be determined, and the long-term effects of stem cell-based therapy on clinical outcomes need further exploration.Autologous stem cell-based therapy is effective and safe for improving the healing of chronic lower extremity ulcers and large-scale RCTs are needed to confirm our findings.
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Úlcera de la Pierna/cirugía , Trasplante de Células Madre/métodos , Trasplante Autólogo/métodos , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Cicatrización de HeridasRESUMEN
Our previous study suggested that microtubule network alteration affects the process of glycolysis in cardiomyocytes (CMs) via the regulation of hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known regarding the underlying mechanisms of microtubule network alteration-induced changes of HIF-1α. The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to mediate the ubiquitination of HIF-1α in the nuclear compartment prior to HIF-1α exportation to the cytoplasm, and pVHL dynamic nuclear-cytoplasmic trafficking is indicated to be involved in the process of HIF-1α degradation. In this study, by administering different microtubule-stabilizing and -depolymerizing interventions, we demonstrated that microtubule stabilization promoted pVHL nuclear export and drove the translocation of pVHL to the cytoplasm, while microtubule disruption prevented pVHL nuclear export in hypoxic CMs. Moreover, the ratio between nuclear and cytoplasmic pVHL was associated with HIF-1α regulation. Importantly, microtubule network alteration also affected the subcellular localization of Ran, which was involved in the regulation of pVHL nuclear-cytoplasmic trafficking. The above results suggest that the subcellular translocation of pVHL plays an important role in microtubular structure alteration-induced HIF-1α regulation. Interestingly, Ran is involved in the process of pVHL nuclear-cytoplasmic trafficking following microtubule network alteration in hypoxic CMs.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Microtúbulos/metabolismo , Miocitos Cardíacos/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Espacio Intracelular/metabolismo , Modelos Biológicos , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Ratas , Tubulina (Proteína)/metabolismoRESUMEN
Keratinocyte migration is an early event in the wound healing process. Although we previously found that CD9 downregulation is required for the keratinocyte migration during wound repair, the mechanism of how CD9 expression is regulated remains unclear. Here, we observed the effect of hypoxia (2% O2) on CD9 expression and keratinocyte migration. CD9 expression was downregulated and keratinocyte migration was increased under hypoxic conditions. In addition, CD9 overexpression reversed hypoxia-induced cell migration. We also found that hypoxia activated the p38/MAPK pathway. SB203580, a p38/MAPK inhibitor, increased CD9 expression and inhibited keratinocyte migration under hypoxia, while MKK6 (Glu) overexpression decreased CD9 expression and promoted hypoxic keratinocyte migration. Our results demonstrate that hypoxia regulates CD9 expression and CD9-mediated keratinocyte migration via the p38/MAPK pathway.
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Hipoxia de la Célula/fisiología , Movimiento Celular/fisiología , Tetraspanina 29/biosíntesis , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Queratinocitos/metabolismo , Queratinocitos/fisiología , MAP Quinasa Quinasa 6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Piridinas/farmacología , Tetraspanina 29/genética , Cicatrización de Heridas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Our previous research found that tetraspanin CD9 is downregulated in migrating epidermis during wound healing, and CD9 downregulation contributes to keratinocyte migration via matrix metalloproteinase-9 (MMP-9) activation. However, little is known about the mechanisms involved in CD9-regulated keratinocyte migration and MMP-9 activation. In this study, we revealed that the expressions of integrin subunits ß5 and ß6 were regulated by CD9. Furthermore, CD9 silencing triggered the switch from αvß5 to αvß6 integrin in HaCaT keratinocytes and CD9 overexpression reversed the switch. Importantly, integrin αvß6 functional blocking antibody 10D5 significantly inhibited CD9 silencing-induced keratinocyte migration and MMP-9 activation, suggesting that the switch from αvß5 to αvß6 integrin plays a key role in CD9-regulated cell migration and MMP-9 activation in keratinocytes.
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Antígenos de Neoplasias/metabolismo , Movimiento Celular , Integrinas/metabolismo , Queratinocitos/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores de Vitronectina/metabolismo , Tetraspanina 29/metabolismo , Anticuerpos/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular , Regulación hacia Abajo , Activación Enzimática , Silenciador del Gen , Humanos , Integrinas/inmunología , Tetraspanina 29/deficiencia , Tetraspanina 29/genéticaRESUMEN
Wound healing is a complex but well-orchestrated tissue repair process composed of a series of molecular and cellular events conducted by various types of cells and extracellular matrix. Despite a variety of therapeutic strategies proposed to accelerate the healing of acute and/or chronic wounds over the past few decades, effective treatment of chronic nonhealing wounds still remains a challenge. Due to the recent advances in stem cell research, a dramatic enthusiasm has been drawn to the application of stem cells in regenerative medicine. Both embryonic and adult stem cells have prolonged self-renewal capacity and are able to differentiate into various tissue types. Nevertheless, use of embryonic stem cells is limited, owing to ethical concerns and legal restrictions. Adult stem cells, which could be isolated from bone marrow, umbilical cord blood, adipose tissue, skin and hair follicles,are being explored extensively to facilitate the healing of both acute and chronic wounds. The current article summarizes recent research on various types of stem cell-based strategies applied to improve wound healing. In addition, future directions of stem cell-based therapy in wound healing have also been discussed. Finally, despite its apparent advantages, limitations and challenges of stem cell therapy are discussed.
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Tratamiento Basado en Trasplante de Células y Tejidos , Medicina Regenerativa , Piel/patología , Trasplante de Células Madre , Cicatrización de Heridas , Heridas y Lesiones/patología , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Matriz Extracelular/patología , Femenino , Humanos , Masculino , Medicina Regenerativa/tendencias , Piel/lesiones , Células Madre/patología , Heridas y Lesiones/terapiaRESUMEN
Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.
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Movimiento Celular/fisiología , Queratinocitos/citología , Queratinocitos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Tetraspanina 29/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Células Cultivadas , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Tetraspanina 29/genéticaRESUMEN
BACKGROUND: Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α. METHODS: MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3'UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro. RESULTS: miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3'UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis. CONCLUSION: miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF-1α by binding directly to its 3'UTR.
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Quemaduras/genética , Movimiento Celular/genética , Quimiocina CXCL12/genética , Silenciador del Gen , Células Madre Mesenquimatosas/patología , MicroARNs/genética , Cicatrización de Heridas/genética , Animales , Quemaduras/patología , Quemaduras/fisiopatología , Quimiocina CXCL12/deficiencia , Biología Computacional , Regulación hacia Abajo , Femenino , Calor , Masculino , Ratones , Piel/patologíaRESUMEN
BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) play a crucial role in tissue repair. Their role in thermal burn wound regeneration and the relevant mechanism, however, is rarely studied. METHODS: BM-MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice. Twenty-one days later, the female mice were inflicted with burn wounds. The size of the burned area was measured by an in vivo fluorescence imaging system, and BM-MSC chemotaxis and epithelialization were estimated by fluorescence in situ hybridization and immunofluorescence technology. The expression of CXCL12 and CXCR4 in the wound margin was detected by enzyme-linked immunosorbent assay and immunohistochemistry. The importance of CXCL12/CXCR4 signaling in BM-MSC chemotaxis was further estimated by blocking CXCR4 in vivo and in vitro. RESULTS: In vivo imaging results showed that BM-MSCs migrated to the injured margins. Fluorescence in situ hybridization and immunofluorescence technology revealed that Y chromosome-positive cells derived from green fluorescent protein transgenic mice were detected to be colocalized with keratin protein. Enzyme-linked immunosorbent assay revealed increased levels of CXCL12 and CXCR4 protein in the wound sites of BM-MSC-treated chimeric mice after burn. Immunohistochemistry also disclosed that CXCL12 levels were elevated at postburn day 7 compared with day 0. Furthermore, pretreatment of the BM-MSCs with the CXCR4 antagonist AMD3100 significantly inhibited the mobilization of BM-MSCs in vitro and in vivo, which attenuated wound closure. CONCLUSION: BM-MSC migration to the burned margins promotes the epithelialization of the wound, and mobilization of BM-MSCs is mediated by CXCL12/CXCR4 signaling.
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Quemaduras/metabolismo , Quimiocina CXCL12/metabolismo , Células Madre Mesenquimatosas/fisiología , Repitelización , Receptores CXCR4/metabolismo , Animales , Quimiotaxis , Quimera , Células Epidérmicas , Femenino , Proteínas Fluorescentes Verdes , Folículo Piloso/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Mitochondrial damage plays an important role in mediating postburn cardiac injury. To elucidate the pivotal mitochondrial proteins and pathways underlying postburn cardiac injury, mitochondria were purified from control and postburn rat hearts. 2-dimensional gel electrophoresis (2-DE) and HPLC-chip-MS/MS analyses revealed 9 differentially expressed proteins, 3 of which were further validated by Western blotting. The differential expression of these mitochondrial proteins was accompanied by increased levels of oxidative cardiac damage and decreased levels of cardiac output. One of the differentially expressed proteins, mitochondria translation elongation factor Tu (EF-Tumt), was hypothesized to contribute crucially to postburn oxidative cardiac damage. The small interfering RNA (siRNA)-mediated downregulation of EF-Tumt in cultured rat cardiomyocytes increased reactive oxygen species (ROS) generation and protein carbonyl levels, and led to cell damage. The potential pathway of this process was associated with respiratory chain complex I deficiency. Together, these results demonstrate the mitochondrial responses to severe burn, and indicate a pathway by which decreased EF-Tumt expression mediates oxidative damage in postburn myocardium.
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Quemaduras/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Animales , Quemaduras/patología , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica , Masculino , Mitocondrias Cardíacas/patología , Miocardio/patología , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A major challenge in cardiovascular regenerative medicine is the development of novel therapeutic strategies to restore the function of cardiac muscle in the failing heart. The heart has historically been regarded as a terminally differentiated organ that does not have the potential to regenerate. This concept has been updated by the discovery of cardiac stem and progenitor cells that reside in the adult mammalian heart. Whereas diverse types of adult cardiac stem or progenitor cells have been described, we still do not know whether these cells share a common origin. A better understanding of the physiology of cardiac stem and progenitor cells should advance the successful use of regenerative medicine as a viable therapy for heart disease. In this review, we summarize current knowledge of the various adult cardiac stem and progenitor cell types that have been discovered. We also review clinical trials presently being undertaken with adult stem cells to repair the injured myocardium in patients with coronary artery disease.
Asunto(s)
Miocitos Cardíacos/fisiología , Medicina Regenerativa/métodos , Células Madre/fisiología , Animales , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones , Miocitos Cardíacos/trasplanteRESUMEN
BACKGROUND: Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn. METHODS: Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function. RESULTS: Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function. CONCLUSION: Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction.
Asunto(s)
Autofagia , Quemaduras/patología , Miocardio/patología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Beclina-1 , Biomarcadores/metabolismo , Compuestos de Bifenilo/farmacología , Quemaduras/fisiopatología , Enalaprilato/farmacología , Técnica del Anticuerpo Fluorescente , Pruebas de Función Cardíaca/efectos de los fármacos , Técnicas In Vitro , Losartán/farmacología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Compuestos Onio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: Nicotinamide plays a protective role in hypoxia-induced cardiomyocyte dysfunction. However, the underlying molecular mechanisms remain poorly understood. The purpose of this study was to investigate these and the effect of nicotinamide pretreatment on hypoxic cardiomyocytes. METHODS: Cultured rat cardiomyocytes were pretreated with nicotinamide, subjected to hypoxia for 6 h, and then cell necrosis and apoptosis were examined. The effects of nicotinamide pretreatment on hypoxia-induced reactive oxygen species (ROS) formation, antioxidant enzyme expression, nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) levels, adenosine triphosphate (ATP) production and mitochondrial membrane potential were tested to elucidate the underlying mechanisms. RESULTS: Based on the findings that nicotinamide treatment decreased protein expression of receptor-interacting protein (RIP; a marker for cell necrosis) and cleaved caspase-3 (CC3; a marker for cell apoptosis) in normoxic cardiomyocytes, we found that it dramatically reduced hypoxia-induced necrosis and apoptosis in cardiomyocytes. The underlying mechanisms of these effects are associated with the fact that it increased protein expression of superoxide dismutase and catalase, increased intracellular levels of NAD(+) and ATP concentration, decreased mitochondrial ROS generation and prevented the loss of mitochondrial membrane potential. CONCLUSION: All of these results indicate that nicotinamide pretreatment protects cardiomyocytes by improving mitochondrial stress. Our study provides a new clue for the utilization of nicotinamide in therapies for ischemic heart disease.
Asunto(s)
Miocitos Cardíacos/efectos de los fármacos , Niacinamida/farmacología , Sustancias Protectoras/farmacología , Complejo Vitamínico B/farmacología , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Hipoxia de la Célula/fisiología , Células Cultivadas , L-Lactato Deshidrogenasa/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Necrosis/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.