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1.
FASEB J ; 35(11): e22017, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34699642

RESUMO

Cellular interactions between endothelial cells and macrophages regulate macrophage localization and phenotype, but the mechanisms underlying these interactions are poorly understood. Here we explored the role of sialoglycans on lymphatic endothelial cells (LEC) in interactions with macrophage-expressed Siglec-1 (CD169). Lectin-binding assays and mass spectrometric analyses revealed that LEC from human skin express more sialylated glycans than the corresponding blood endothelial cells. Higher amounts of sialylated and/or sulfated glycans on LEC than BEC were consistently observed in murine skin, lung and lymph nodes. The floor LEC of the subcapsular sinus (SCS) in murine lymph nodes (LN) displayed sialylated glycans at particularly high densities. The sialoglycans of LN LEC were strongly bound by Siglec-1. Such binding plays an important role in the localization of Siglec-1+ LN-SCS macrophages, as their numbers are strongly reduced in mice expressing a Siglec-1 mutant that is defective in sialoglycan binding. The residual Siglec-1+ macrophages are less proliferative and have a more anti-inflammatory phenotype. We propose that the densely clustered, sialylated glycans on the SCS floor LEC are a key component of the macrophage niche, providing anchorage for the Siglec-1+ LN-SCS macrophages.


Assuntos
Células Endoteliais/metabolismo , Linfonodos/metabolismo , Macrófagos/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Pele/metabolismo , Animais , Células CHO , Cricetulus , Células Endoteliais/citologia , Humanos , Linfonodos/citologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Pele/citologia
2.
Immunity ; 54(10): 2305-2320.e11, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34508661

RESUMO

Langerhans cells (LCs) play a pivotal role in skin homeostasis, and the heterogeneity of LCs has long been considered. In this study, we have identified two steady-state (LC1 and LC2) and two activated LC subsets in the epidermis of human skin and in LCs derived from CD34+ hemopoietic stem cells (HSC-LCs) by utilizing single-cell RNA sequencing and mass cytometry. Analysis of HSC-LCs at multiple time-points during differentiation revealed that EGR1 and Notch signaling were among the top pathways regulating the bifurcation of LC1 and LC2. LC1 were characterized as classical LCs, mainly related to innate immunity and antigen processing. LC2 were similar to monocytes or myeloid dendritic cells, involving in immune responses and leukocyte activation. LC1 remained stable under inflammatory microenvironment, whereas LC2 were prone to being activated and demonstrated elevated expression of immuno-suppressive molecules. We revealed distinct human LC subsets that require different developmental regulation and orchestrate reciprocal functions.


Assuntos
Diferenciação Celular/imunologia , Células de Langerhans/citologia , Células de Langerhans/imunologia , Pele/citologia , Pele/imunologia , Apresentação do Antígeno/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunidade Inata/imunologia
3.
Cells ; 10(9)2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34572048

RESUMO

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.


Assuntos
Diferenciação Celular , Linhagem da Célula , Corpos Embrioides/citologia , Rim/citologia , Células-Tronco Pluripotentes/citologia , Pele/citologia , Inativação do Cromossomo X , Corpos Embrioides/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Rim/metabolismo , Masculino , Células-Tronco Pluripotentes/metabolismo , Pele/metabolismo
4.
Biochem Biophys Res Commun ; 577: 64-70, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34507067

RESUMO

To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.


Assuntos
Expressão Gênica , Luciferases/genética , Medições Luminescentes/métodos , Proteínas Circadianas Period/genética , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Cabelo/metabolismo , Luciferases/metabolismo , Medições Luminescentes/instrumentação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Movimento/fisiologia , Proteínas Circadianas Period/metabolismo , Reprodutibilidade dos Testes , Couro Cabeludo/metabolismo , Pele/citologia , Pele/metabolismo , Vibrissas/metabolismo
5.
Sci Rep ; 11(1): 17916, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504144

RESUMO

Exposure of cells or biological tissues to high-power pulses of terahertz (THz) radiation leads to changes in a variety of intracellular processes. However, the role of heating effects due to strong absorption of THz radiation by water molecules still stays unclear. In this study, we performed numerical modelling in order to estimate the thermal impact on water of a single THz pulse as well as a series of THz pulses. A finite-element (FE) model that provides numerical solutions for the heat conduction equation is employed to compute the temperature increase. A simple expression for temperature estimation in the center of the spot of THz radiation is presented for given frequency and fluence of the THz pulse. It has been demonstrated that thermal effect is determined by either the average power of radiation or by the fluence of a single THz pulse depending on pulse repetition rate. Human dermal fibroblasts have been exposed to THz pulses (with an energy of [Formula: see text] and repetition rate of 100 Hz) to estimate the thermal effect. Analysis of heat shock proteins expression has demonstrated no statistically significant difference ([Formula: see text]) between control and experimental groups after 3 h of irradiation.


Assuntos
Fibroblastos , Proteínas de Choque Térmico/metabolismo , Temperatura Alta/efeitos adversos , Pele , Radiação Terahertz/efeitos adversos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Pele/citologia , Pele/metabolismo
6.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361066

RESUMO

Ceramides, a class of sphingolipids containing a backbone of sphingoid base, are the most important and effective structural component for the formation of the epidermal permeability barrier. While ceramides comprise approximately 50% of the epidermal lipid content by mass, the content is substantially decreased in certain inflammatory skin diseases, such as atopic dermatitis (AD), causing improper barrier function. It is widely accepted that the endocannabinoid system (ECS) can modulate a number of biological responses in the central nerve system, prior studies revealed that activation of endocannabinoid receptor CB1, a key component of ECS, triggers the generation of ceramides that mediate neuronal cell fate. However, as the impact of ECS on the production of epidermal ceramide has not been studied, we here investigated whether the ECS stimulates the generation of epidermal ceramides in an IL-4-treated in vitro model of skin inflammation using N-palmitoyl serinol (PS), an analog of the endocannabinoid N-palmitoyl ethanolamine. Accordingly, an IL-4-mediated decrease in cellular ceramide levels was significantly stimulated in human epidermal keratinocytes (KC) following PS treatment through both de novo ceramide synthesis- and sphingomyelin hydrolysis-pathways. Importantly, PS selectively increases ceramides with long-chain fatty acids (FAs) (C22-C24), which mainly account for the formation of the epidermal barrier, through activation of ceramide synthase (CerS) 2 and Cer3 in IL-4-mediated inflamed KC. Furthermore, blockade of cannabinoid receptor CB1 activation by AM-251 failed to stimulate the production of total ceramide as well as long-chain ceramides in response to PS. These studies demonstrate that an analog of endocannabinoid, PS, stimulates the generation of specific ceramide species as well as the total amount of ceramides via the endocannabinoid receptor CB1-dependent mechanism, thereby resulting in the enhancement of epidermal permeability barrier function.


Assuntos
Ceramidas/metabolismo , Inflamação/metabolismo , Queratinócitos/metabolismo , Propanolaminas/farmacologia , Propilenoglicóis/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Pele/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Propanolaminas/química , Propilenoglicóis/química , Pele/citologia , Pele/efeitos dos fármacos
7.
Carbohydr Polym ; 270: 117916, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34364636

RESUMO

A novel brush-like poly(2-aminoethyl methacrylate) (PAEMA) was grafted onto chitosan (CS) through gamma radiation-induced polymerization. The copolymer (CS-g-PAEMA) was used to prepare a sodium acetate leached poly(urethane-urea) scaffold. The above derivatives were developed, synthesized, and characterized to meet the specific characteristics of biomaterials. The results revealed that this method is an easy and successful route for grafting PAEMA onto CS. The feasibility of preparing a CS-g-PAEMA polyurethane foam was confirmed by mechanical, morphometric, spectroscopic, and cytotoxic studies. The scaffold showed high biocompatibility both in vitro and in vivo. The first experiment proved that CS-based polyurethane efficiently allows the dynamic culturing of human fibroblast cells. Additionally, an in vivo study in a murine model indicated a complete integration of the scaffold to surrounding subcutaneous tissue as supported by the histological and histochemical assessments. The aforementioned results support the use of CS-g-PAEMA poly(saccharide-urethane) as a model of in vitro-engineered skin.


Assuntos
Quitosana/química , Metacrilatos/química , Polímeros/química , Poliuretanos/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Fibroblastos/citologia , Raios gama , Humanos , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Polimerização , Pele/citologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
8.
Sci Rep ; 11(1): 16164, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373593

RESUMO

Using the skin tissue engineering approach is a way to help the body to recover its lost skin in cases that the spontaneous healing process is either impossible or inadequate, such as severe wounds or burns. In the present study, chitosan/gelatin-based scaffolds containing 0.25, 0.5, 0.75, and 1% allantoin were created to improve the wounds' healing process. EDC and NHS were used to cross-link the samples, which were further freeze-dried. Different in-vitro methods were utilized to characterize the specimens, including SEM imaging, PBS absorption and degradation tests, mechanical experiments, allantoin release profile assessment, antibacterial assay, and cell viability and adhesion tests. The results indicated that the scaffolds' average pore sizes were approximately in the range of 390-440 µm, and their PBS uptake amounts were about 1000% to 1250% after being soaked in PBS for 24 h. Around 70% of the specimens were degraded in 6 days, but they were not fully degraded after 21 days. Besides, the samples showed antibacterial activity against S. aureus and E. coli bacteria. In general, the MTT cell viability test indicated that the cells' density increased slightly or remained the same during the experiment. SEM images of cells seeded on the scaffolds indicated appropriate properties of the scaffolds for cell adhesion.


Assuntos
Alantoína/análise , Transplante de Pele/métodos , Pele/lesões , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Antibacterianos/análise , Antibacterianos/farmacologia , Materiais Biocompatíveis/análise , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Quitosana , Escherichia coli/efeitos dos fármacos , Gelatina , Humanos , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Porosidade , Medicina Regenerativa , Reologia , Pele/citologia , Fenômenos Fisiológicos da Pele , Staphylococcus aureus/efeitos dos fármacos , Cicatrização
9.
Biomolecules ; 11(8)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34439879

RESUMO

Cell adhesion molecules (CAMs) of the cadherin, integrin, immunoglobulin, and selectin protein families are indispensable for the formation and maintenance of multicellular tissues, especially epithelia. In the epidermis, they are involved in cell-cell contacts and in cellular interactions with the extracellular matrix (ECM), thereby contributing to the structural integrity and barrier formation of the skin. Bulk and single cell RNA sequencing data show that >170 CAMs are expressed in the healthy human skin, with high expression levels in melanocytes, keratinocytes, endothelial, and smooth muscle cells. Alterations in expression levels of CAMs are involved in melanoma propagation, interaction with the microenvironment, and metastasis. Recent mechanistic analyses together with protein and gene expression data provide a better picture of the role of CAMs in the context of skin physiology and melanoma. Here, we review progress in the field and discuss molecular mechanisms in light of gene expression profiles, including recent single cell RNA expression information. We highlight key adhesion molecules in melanoma, which can guide the identification of pathways and strategies for novel anti-melanoma therapies.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Melanócitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/metabolismo , Comunicação Celular , Humanos , Melanócitos/citologia , Melanócitos/patologia , Pele/citologia , Pele/patologia , Microambiente Tumoral
10.
Biomed Res Int ; 2021: 1340281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336999

RESUMO

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.


Assuntos
Células-Tronco Mesenquimais/citologia , Pele/citologia , Vitrificação , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Derme/citologia , Cães , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
11.
Molecules ; 26(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34361611

RESUMO

UV-B and IR-A radiation are important inducers of biological changes in skin involving ROS generation. The overloading of antioxidant defense mechanisms by ROS production could lead to photoaging and photocarcinogenesis processes. Various traditional usages are reported for Aralia nudicaulis L. extracts, including treatment of dermatological disorders. Antioxidant and anti-inflammatory properties have already been reported for other Aralia species possibly due to the presence of phenolic compounds. However, the phenolic composition and the potential activity of A. nudicaulis rhizomes extract against oxidative stress and UV/IR damages have not been investigated. The main aims of this study were to prepare a fraction enriched in phenolic compounds (FEPC) from A. nudicaulis rhizomes, to identify its major phenolic compounds and to assess its potential for protective effects against oxidative stress induced by UV-B, IR-A or inflammation. A quantitative LC-MS study of FEPC shows that chlorogenic, caffeic and protocatechuic acids are the main phenolic compounds present, with concentrations of 15.6%, 15.3% and 4.8% of the total composition, respectively. With a validated analytical method, those compounds were quantified over different stages of the growing period. As for biological potential, first this extract demonstrates antioxidant and anti-inflammatory activities. Furthermore, ROS generation induced by IR-A and UV-B were strongly inhibited by A. nudicaulis extract, suggesting that Aralia nudicaulis L. rhizome extract could protect dermal cells against oxidative stress induced by UV-B and IR-A.


Assuntos
Antioxidantes/farmacologia , Aralia/química , Fibroblastos/efeitos dos fármacos , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Pele/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Linhagem Celular , Fibroblastos/citologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rizoma/química , Pele/citologia
12.
Biomolecules ; 11(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34356642

RESUMO

Collagen and proteoglycans work in unison in the ECM to bear loads, store elastic energy and then dissipate excess energy to avoid tissue fatigue and premature mechanical failure. While collagen fibers store elastic energy by stretching the flexible regions in the triple helix, they do so by lowering their free energy through a reduction in the entropy and a decrease in charge-charge repulsion. Entropic increases occur when the load is released that drive the reversibility of the process and transmission of excess energy. Energy is dissipated by sliding of collagen fibrils by each other with the aid of decorin molecules that reside on the d and e bands of the native D repeat pattern. Fluid flow from the hydration layer associated with the decorin and collagen fibrils hydraulically dissipates energy during sliding. The deformation is reversed by osmotic forces that cause fluid to reform a hydration shell around the collagen fibrils when the loads are removed. In this paper a model is presented describing the organization of collagen fibers in the skin and cell-collagen mechanical relationships that exist based on non-invasive measurements made using vibrational optical coherence tomography. It is proposed that under external stress, collagen fibers form a tensional network in the plane of the skin. Collagen fiber tension along with forces generated by fibroblasts exerted on collagen fibers lead to an elastic modulus that is almost uniform throughout the plane of the skin. Tensile forces acting on cells and tissues may provide a baseline for stimulation of normal mechanotransduction. We hypothesize that during aging, changes in cellular metabolism, cell-collagen interactions and light and UV light exposure cause down regulation of mechanotransduction and tissue metabolism leading to tissue atrophy.


Assuntos
Matriz Extracelular/química , Colágenos Fibrilares , Proteoglicanas , Pele/citologia , Tomografia de Coerência Óptica/métodos , Adulto , Idoso , Fenômenos Biomecânicos , Módulo de Elasticidade , Feminino , Colágenos Fibrilares/química , Colágenos Fibrilares/metabolismo , Humanos , Masculino , Mecanotransdução Celular , Proteoglicanas/química , Proteoglicanas/metabolismo , Fenômenos Fisiológicos da Pele , Adulto Jovem
13.
Nucleic Acids Res ; 49(15): 8714-8731, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379776

RESUMO

Microhomology-mediated break-induced replication (MMBIR) is a DNA repair pathway initiated by polymerase template switching at microhomology, which can produce templated insertions that initiate chromosomal rearrangements leading to neurological and metabolic diseases, and promote complex genomic rearrangements (CGRs) found in cancer. Yet, how often templated insertions accumulate from processes like MMBIR in genomes is poorly understood due to difficulty in directly identifying these events by whole genome sequencing (WGS). Here, by using our newly developed MMBSearch software, we directly detect such templated insertions (MMB-TIs) in human genomes and report substantial differences in frequency and complexity of MMB-TI events between normal and cancer cells. Through analysis of 71 cancer genomes from The Cancer Genome Atlas (TCGA), we observed that MMB-TIs readily accumulate de novo across several cancer types, with particularly high accumulation in some breast and lung cancers. By contrast, MMB-TIs appear only as germline variants in normal human fibroblast cells, and do not accumulate as de novo somatic mutations. Finally, we performed WGS on a lung adenocarcinoma patient case and confirmed MMB-TI-initiated chromosome fusions that disrupted potential tumor suppressors and induced chromothripsis-like CGRs. Based on our findings we propose that MMB-TIs represent a trigger for widespread genomic instability and tumor evolution.


Assuntos
Reparo do DNA , Neoplasias/genética , Adenocarcinoma de Pulmão/genética , Fibroblastos , Genes Supressores de Tumor , Genoma Humano , Instabilidade Genômica , Humanos , Neoplasias Pulmonares/genética , Mutagênese Insercional , Pele/citologia , Software
14.
Cells ; 10(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34360001

RESUMO

Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues.


Assuntos
Envelhecimento/fisiologia , Epitélio/fisiologia , Queratinócitos/citologia , Mecanotransdução Celular/fisiologia , Pele Artificial , Pele/citologia , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Reatores Biológicos , Comunicação Celular , Elasticidade , Embrião de Mamíferos , Epitélio/anatomia & histologia , Queratinócitos/fisiologia , Camundongos , Ratos , Resistência à Tração , Viscosidade
15.
Cells ; 10(8)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34440869

RESUMO

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.


Assuntos
Fibroblastos/citologia , Cultura Primária de Células/métodos , Pele/citologia , Animais , Biomarcadores/metabolismo , Separação Celular , Sobrevivência Celular , Endopeptidases/metabolismo , Fibroblastos/metabolismo , Lagomorpha , Tripsina/metabolismo
17.
Nat Commun ; 12(1): 4404, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285225

RESUMO

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-ß (TGFß) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFß induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFß on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFß-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFß signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.


Assuntos
Autofagia/genética , Epigênese Genética , Histona Acetiltransferases/metabolismo , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Biópsia , Estudos de Casos e Controles , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Fibroblastos , Fibrose , Técnicas de Inativação de Genes , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Células NIH 3T3 , Cultura Primária de Células , Receptores de Fatores de Crescimento Transformadores beta , Transdução de Sinais/genética , Pele/citologia , Pele/patologia , Proteína Smad3/metabolismo , Adulto Jovem
18.
J Photochem Photobiol B ; 221: 112255, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34271412

RESUMO

Skin is the largest body organ and can be affected by several factors, such as ultraviolet (UV) radiation. UV radiation is subdivided in UVA, UVB and UVC according to the radiation wavelength. UVC radiation does not cross the ozone layer; UVB cause DNA damage and is closely related to carcinogenesis; UVA radiation penetrates deeply into the skin, reaching epidermis and dermis and is considered the main promoter of skin aging, known as photoaging. In order to understand photoaging mechanisms and propose efficient therapies, several photoaging study models have been developed, each with benefits and limitations, but most of them use very high doses of UVA radiation, which is not compatible with our daily sun exposure. The objective of this work was to develop a human ex vivo photoaging model induced by UVA exposure compatible to a summer in Brazil. For this, human skin fragments were obtained from healthy donors who underwent otoplasty surgery and skin explants were prepared and placed in plates, with the epidermis facing upwards. Skin explants were exposed to UVA at 16 J/cm2 carried out by protocols of 2 or 4 exposures. Results showed an increase of oxidative damage, inflammatory cells, collagenolytic and elastolytic MMPs expression as well as a decrease of elastin expression, suggesting that the experimental model based on skin explants is able to evaluate UVA-induced aging in human skin.


Assuntos
Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta , Brasil , Sobrevivência Celular/efeitos da radiação , Humanos , Estresse Oxidativo/efeitos da radiação , Estações do Ano , Pele/citologia , Pele/patologia , Pele/efeitos da radiação
19.
Methods Mol Biol ; 2319: 61-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331243

RESUMO

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.


Assuntos
Células Endoteliais/citologia , Imuno-Histoquímica/métodos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Pele/citologia , Coloração e Rotulagem/métodos , Animais , Células Endoteliais/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Pele/crescimento & desenvolvimento , Pele/metabolismo
20.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203413

RESUMO

To date, placental trophoblasts have been of interest in the fields of obstetrics and gynecology, mainly due to their involvement in the formation of a connection between the mother and fetus that aids in placental development and fetal survival. However, the regenerative capacities of trophoblasts for application in regenerative medicine and tissue engineering are poorly understood. Here, we aim to determine the skin regeneration and anti-aging capacities of trophoblast-derived conditioned medium (TB-CM) and exosomes (TB-Exos) using human normal dermal fibroblasts (HNDFs). TB-CM and TB-Exos treatments significantly elevated the migration and proliferation potencies of HNDF cells in a dose- and time-dependent manner. When RNA sequencing (RNA-seq) was used to investigate the mechanism underlying TB-CM-induced cell migration on scratch-wounded HNDFs, the increased expression of genes associated with C-X-C motif ligand (CXCL) chemokines, toll-like receptors, and nuclear factor-kappa B (NF-κB) signaling was observed. Furthermore, treatment of intrinsically/extrinsically senescent HNDFs with TB-CM resulted in an enhanced rejuvenation of HNDFs via both protection and restoration processes. Gene expression of extracellular matrix components in the skin dermis significantly increased in TB-CM- and TB-Exos-treated HNDFs. These components are involved in the TB-CM and Exo-mediated regeneration and anti-aging of HNDFs. Thus, this study demonstrated the regenerative and anti-aging efficacies of trophoblast-derived secretomes, suggesting their potential for use in interventions for skin protection and treatment.


Assuntos
Fibroblastos/citologia , Pele/citologia , Trofoblastos/citologia , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos da radiação , Meios de Cultivo Condicionados/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Trofoblastos/efeitos dos fármacos , Trofoblastos/efeitos da radiação , Raios Ultravioleta
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