RESUMEN
Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in â¼86% of familial and â¼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.
Asunto(s)
Neurilemoma , Neurofibromatosis , Cromosomas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neurilemoma/genética , Neurilemoma/patología , Neurofibromatosis/genética , Proteína SMARCB1/genética , Factores de Transcripción/genéticaRESUMEN
Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).
Asunto(s)
Proliferación Celular/efectos de los fármacos , Oligopéptidos/farmacología , Piel/patología , Cicatrización de Heridas , Albúminas/metabolismo , Animales , Basófilos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Quimiotaxis/efectos de los fármacos , Citocinas/metabolismo , Metilación de ADN/efectos de los fármacos , Oído/patología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HaCaT/citología , Células HaCaT/efectos de los fármacos , Humanos , Inyecciones Subcutáneas , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligopéptidos/sangre , Oligopéptidos/química , Oligopéptidos/metabolismo , Estabilidad Proteica/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Adipose-derived mesenchymal stromal cells (AD-MSCs) are an essential issue in modern medicine. Extensive preclinical and clinical studies have shown that mesenchymal stromal/stem cells, including AD-MSCs, have specific properties (ability to differentiate into other cells, recruitment to the site of injury) of particular importance in the regenerative process. Ongoing research aims to elucidate factors supporting AD-MSC culture and differentiation in vitro. Angiopoietin-like proteins (ANGPTLs), known for their pleiotropic effects in lipid and glucose metabolism, may play a significant role in this context. Regeneration is a complex and dynamic process controlled by many factors. ANGPTL6 (Angiopoietin-related growth factor, AGF), among many activities modulated the biological activity of stem cells. This study examined the influence of synthesized AGF-derived peptides, designated as AGF9 and AGF27, on AD-MSCs. AGF9 and AGF27 enhanced the viability and migration of AD-MSCs and acted as a chemotactic factor for these cells. AGF9 stimulated chondrogenesis and lipid synthesis during AD-MSCs differentiation, influenced AD-MSCs cytokine secretion and modulated transcriptome for such basic cell activities as migration, transport of molecules, and apoptosis. The ability of AGF9 to modulate the biological activity of AD-MSCs warrants the consideration of this peptide a noteworthy therapeutic agent that deserves further investigation for applications in regenerative medicine.
Asunto(s)
Tejido Adiposo , Proteínas Similares a la Angiopoyetina , Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Humanos , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Diferenciación Celular/efectos de los fármacos , Proteínas Similares a la Angiopoyetina/metabolismo , Condrogénesis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Péptidos/farmacología , Movimiento Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Citocinas/metabolismoRESUMEN
Objective: This study evaluated the use of novel peptides derived from platelet-derived growth factor (PDGF-BB) as potential wound healing stimulants. One of the compounds (named PDGF2) was subjected for further research after cytotoxicity and proliferation assays on human skin cells. Further investigation included evaluation of: migration and chemotaxis of skin cells, immunological and allergic safety, the transcriptional analyses of adipose-derived stem cells (ASCs) and dermal fibroblasts stimulated with PDGF2, and the use of dorsal skin wound injury model to evaluate the effect of wound healing in mice. Approach: Colorimetric lactate dehydrogenase and tetrazolium assays were used to evaluate the cytotoxicity and the effect on proliferation. PDGF2 effect on migration and chemotaxis was also checked. Immunological safety and allergic potential were evaluated with a lymphocyte activation and basophil activation test. Transcriptional profiles of ASCs and primary fibroblasts were assessed after stimulation with PDGF2. Eight-week-old BALB/c female mice were used for dorsal skin wound injury model. Results: PDGF2 showed low cytotoxicity, pro-proliferative effects on human skin cells, high immunological safety, and accelerated wound healing in mouse model. Furthermore, transcriptomic analysis of ASCs and fibroblasts revealed the activation of processes involved in wound healing and indicated its safety. Innovation: A novel peptide derived from PDGF-BB was proved to be safe drug candidate in wound healing. We also present a multifaceted in vitro model for the initial screening of new compounds that may be potentially useful in wound healing stimulation. Conclusion: The results show that peptide derived from PDGF-BB is a promising drug candidate for wound treatment.
Asunto(s)
Tejido Adiposo/citología , Becaplermina/farmacología , Fibroblastos/efectos de los fármacos , Células Madre/citología , Cicatrización de Heridas/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Quimiotaxis/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Preparaciones Farmacéuticas , Proteínas Recombinantes , Piel/citología , Células Madre/metabolismoRESUMEN
BACKGROUND: Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity. METHODS: We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice. FINDINGS: We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2⯱â¯9.4% in zebularine-treated and by 43.6⯱â¯15.4% in control mice (pâ¯<â¯10-30). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues. INTERPRETATION: This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs.
Asunto(s)
Citidina/análogos & derivados , Epigénesis Genética/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , Animales , Biomarcadores , Proliferación Celular/efectos de los fármacos , Islas de CpG , Citidina/farmacología , Metilación de ADN/efectos de los fármacos , Pabellón Auricular/efectos de los fármacos , Pabellón Auricular/lesiones , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Medicina Regenerativa , Tretinoina/farmacologíaRESUMEN
Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.