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1.
Sci Rep ; 11(1): 17926, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504132

RESUMO

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal-epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1-/- mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1-/- mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.


Assuntos
Derme/metabolismo , Epiderme/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Transdução de Sinais/genética , Tendões/metabolismo , Animais , Células Cultivadas , Tecido Conjuntivo/metabolismo , Desenvolvimento Embrionário/genética , Proteínas da Matriz Extracelular/genética , Feminino , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Resistência à Tração
2.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201667

RESUMO

Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25-35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to "personalized" skin for the diagnosis or determination of the most effective treatment possible.


Assuntos
Diferenciação Celular , Derme/citologia , Epiderme/fisiologia , Fibrina/metabolismo , Hidrogéis/metabolismo , Queratinócitos/citologia , Tecidos Suporte/química , Proliferação de Células , Células Cultivadas , Derme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Hidrogéis/química , Queratinócitos/metabolismo , Pele/citologia , Pele/metabolismo , Engenharia Tecidual
3.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298962

RESUMO

We studied CD34+ stromal cells/telocytes (CD34+SCs/TCs) in pathologic skin, after briefly examining them in normal conditions. We confirm previous studies by other authors in the normal dermis regarding CD34+SC/TC characteristics and distribution around vessels, nerves and cutaneous annexes, highlighting their practical absence in the papillary dermis and presence in the bulge region of perifollicular groups of very small CD34+ stromal cells. In non-tumoral skin pathology, we studied examples of the principal histologic patterns in which CD34+SCs/TCs have (1) a fundamental pathophysiological role, including (a) fibrosing/sclerosing diseases, such as systemic sclerosis, with loss of CD34+SCs/TCs and presence of stromal cells co-expressing CD34 and αSMA, and (b) metabolic degenerative processes, including basophilic degeneration of collagen, with stromal cells/telocytes in close association with degenerative fibrils, and cutaneous myxoid cysts with spindle-shaped, stellate and bulky vacuolated CD34+ stromal cells, and (2) a secondary reactive role, encompassing dermatitis-e.g., interface (erythema multiforme), acantholytic (pemphigus, Hailey-Hailey disease), lichenoid (lichen planus), subepidermal vesicular (bullous pemphigoid), psoriasiform (psoriasis), granulomatous (granuloma annulare)-vasculitis (leukocytoclastic and lymphocytic vasculitis), folliculitis, perifolliculitis and inflammation of the sweat and sebaceous glands (perifolliculitis and rosacea) and infectious dermatitis (verruca vulgaris). In skin tumor and tumor-like conditions, we studied examples of those in which CD34+ stromal cells are (1) the neoplastic component (dermatofibrosarcoma protuberans, sclerotic fibroma and solitary fibrous tumor), (2) a neoplastic component with varying presentation (fibroepithelial polyp and superficial myxofibrosarcoma) and (3) a reactive component in other tumor/tumor-like cell lines, such as those deriving from vessel periendothelial cells (myopericytoma), epithelial cells (trichoepithelioma, nevus sebaceous of Jadassohn and seborrheic keratosis), Merkel cells (Merkel cell carcinoma), melanocytes (dermal melanocytic nevi) and Schwann cells (neurofibroma and granular cell tumor).


Assuntos
Antígenos CD34/metabolismo , Dermatite/metabolismo , Derme/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Telócitos/metabolismo , Animais , Dermatite/patologia , Derme/patologia , Humanos , Neoplasias Cutâneas/patologia , Telócitos/patologia
4.
Molecules ; 26(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299649

RESUMO

Cosmetics has recently focused on biobased skin-compatible materials. Materials from natural sources can be used to produce more sustainable skin contact products with enhanced bioactivity. Surface functionalization using natural-based nano/microparticles is thus a subject of study, aimed at better understanding the skin compatibility of many biopolymers also deriving from biowaste. This research investigated electrospray as a method for surface modification of cellulose tissues with chitin nanofibrils (CNs) using two different sources-namely, vegetable (i.e., from fungi), and animal (from crustaceans)-and different solvent systems to obtain a biobased and skin-compatible product. The surface of cellulose tissues was uniformly decorated with electrosprayed CNs. Biological analysis revealed that all treated samples were suitable for skin applications since human dermal keratinocytes (i.e., HaCaT cells) successfully adhered to the processed tissues and were viable after being in contact with released substances in culture media. These results indicate that the use of solvents did not affect the final cytocompatibility due to their effective evaporation during the electrospray process. Such treatments did not also affect the characteristics of cellulose; in addition, they showed promising anti-inflammatory and indirect antimicrobial activity toward dermal keratinocytes in vitro. Specifically, cellulosic substrates decorated with nanochitins from shrimp showed strong immunomodulatory activity by first upregulating then downregulating the pro-inflammatory cytokines, whereas nanochitins from mushrooms displayed an overall anti-inflammatory activity via a slight decrement of the pro-inflammatory cytokines and increment of the anti-inflammatory marker. Electrospray could represent a green method for surface modification of sustainable and biofunctional skincare products.


Assuntos
Agaricales/química , Celulose/farmacologia , Quitina/farmacologia , Cosméticos/farmacologia , Derme/metabolismo , Queratinócitos/metabolismo , Penaeidae/química , Animais , Linhagem Celular , Celulose/química , Quitina/química , Cosméticos/química , Humanos , Nanoestruturas
5.
FASEB J ; 35(8): e21762, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34246197

RESUMO

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cicatriz/metabolismo , Conexina 43/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Peptídeos/farmacologia , Animais , Cicatriz/patologia , Matriz Extracelular/metabolismo , Feminino , Cobaias , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
6.
Biochem Biophys Res Commun ; 571: 14-19, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34298337

RESUMO

Restoration of hair follicle (HF) regenerative capacity is the cornerstone in tissue engineering for the loss of regenerative capacity during in vitro expansion of skin-derived precursors (SKPs). Microenvironmental cues facilitated tissue or organ regeneration offers a potential strategy to overcome this difficulty. In our previous work, plantar dermis matrix homogenate (PD) has been proved to induce sweat glands regeneration both in vivo and in vitro. Here, we found PD also restore regenerative capacity of culture impaired HF spheroids (IHFS). Further, followed by our previous iTRAQ results, the CTHRC1 was identified as a potential regulator in PD facilitated restorative effects in HF regeneration. Knockout of Cthrc1 impaired HF regenerative capacity in spheroids, decreased the diameter of HF in 28 postnatal days mice and shortened invagination of HF bud in 18 days of gestation mice. In IHFS and Cthrc1-/- spheroids, PD partially restored HF regenerative capacity while Cthrc1-/- PD (PDKO) has less or no effect. Taken together, PD is an effective microenvironmental cues for HF regenerative capacity restoration and CTHRC1 played an important role in HF regeneration.


Assuntos
Derme/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Folículo Piloso/metabolismo , Animais , Proteínas da Matriz Extracelular/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073402

RESUMO

The development of scaffolds mimicking the extracellular matrix containing bioactive substances has great potential in tissue engineering and wound healing applications. This study investigates melatonin-a methoxyindole present in almost all biological systems. Melatonin is a bioregulator in terms of its potential clinical importance for future therapies of cutaneous diseases. Mammalian skin is not only a prominent melatonin target, but also produces and rapidly metabolizes the multifunctional methoxyindole to biologically active metabolites. In our methodology, chitosan/collagen (CTS/Coll)-contained biomaterials are blended with melatonin at different doses to fabricate biomimetic hybrid scaffolds. We use rat tail tendon- and Salmo salar fish skin-derived collagens to assess biophysical and cellular properties by (i) Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), (ii) thermogravimetric analysis (TG), (iii) scanning electron microscope (SEM), and (iv) proliferation ratio of cutaneous cells in vitro. Our results indicate that melatonin itself does not negatively affect biophysical properties of melatonin-immobilized hybrid scaffolds, but it induces a pronounced elevation of cell viability within human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), and reference melanoma cells. These results demonstrate that this indoleamine accelerates re-epithelialization. This delivery is a promising technique for additional explorations in future dermatotherapy and protective skin medicine.


Assuntos
Bandagens , Quitosana/química , Colágeno/química , Derme/metabolismo , Epiderme/metabolismo , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Melatonina , Linhagem Celular , Derme/patologia , Avaliação Pré-Clínica de Medicamentos , Epiderme/patologia , Fibroblastos/patologia , Humanos , Queratinócitos/patologia , Melatonina/química , Melatonina/farmacocinética , Melatonina/farmacologia
8.
Dev Biol ; 478: 25-40, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34166654

RESUMO

Skin development and patterning is dependent on factors that regulate the stepwise differentiation of dermal fibroblasts concomitant with dermal-epidermal reciprocal signaling, two processes that are poorly understood. Here we show that dermal EZH2, the methyltransferase enzyme of the epigenetic Polycomb Repressive Complex 2 (PRC2), is a new coordinator of both these processes. Dermal EZH2 activity is present during dermal fibroblast differentiation and is required for spatially restricting Wnt/ß-catenin signaling to reinforce dermal fibroblast cell fate. Later in development, dermal EZH2 regulates the expression of reticular dermal markers and initiation of secondary hair follicles. Embryos lacking dermal Ezh2 have elevated epidermal proliferation and differentiation that can be rescued by small molecule inhibition of retinoic acid (RA) signaling. Together, our study reveals that dermal EZH2 is acting like a rheostat to control the levels of Wnt/ß-catenin and RA signaling to impact fibroblast differentiation cell autonomously and epidermal keratinocyte development non-cell autonomously, respectively.


Assuntos
Derme/citologia , Derme/embriologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epiderme/embriologia , Fibroblastos/citologia , Queratinócitos/citologia , Complexo Repressor Polycomb 2/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Derme/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epiderme/metabolismo , Fibroblastos/metabolismo , Hiperplasia , Queratinócitos/metabolismo , Camundongos , Organogênese , Retinoides/farmacologia , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Tretinoína/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
9.
J Microbiol Biotechnol ; 31(7): 933-941, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34099599

RESUMO

Ginsenoside Rg4 is a rare ginsenoside that is naturally found in ginseng, and exhibits a wide range of biological activities including antioxidant and anti-inflammatory properties in several cell types. The purpose of this study was to use an in vivo model of hair follicle (HF)-mimic based on a human dermal papilla (DP) spheroid system prepared by three-dimensional (3D) culture and to investigate the effect of Rg4 on the hair-inductive properties of DP cells. Treatment of the DP spheroids with Rg4 (20 to 50 µg/ml) significantly increased the viability and size of the DP spheres in a dose-dependent manner. Rg4 also increased the mRNA and protein expression of DP signature genes that are related to hair growth including ALP, BMP2, and VCAN in the DP spheres. Analysis of the signaling molecules and luciferase reporter assays further revealed that Rg4 induces the activation of phosphoinositide 3-kinase (PI3K)/AKT and the inhibitory phosphorylation of GSK3ß, which activates the WNT/ß-catenin signaling pathway. These results correlated with not only the increased nuclear translocation of ß-catenin following the treatment of the DP spheres with Rg4 but also the significant elevation of mRNA expression of the downstream target genes of the WNT/ß-catenin pathway including WNT5A, ß-catenin, and LEF1. In conclusion, these results demonstrated that ginsenoside Rg4 promotes the hair-inductive properties of DP cells by activating the AKT/GSK3ß/ß-catenin signaling pathway in DP spheres, suggesting that Rg4 could be a potential natural therapy for hair growth.


Assuntos
Derme/efeitos dos fármacos , Ginsenosídeos/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Cabelo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Derme/citologia , Derme/metabolismo , Cabelo/crescimento & desenvolvimento , Folículo Piloso/citologia , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Esferoides Celulares , Proteínas Wnt/metabolismo
10.
Sci Rep ; 11(1): 12225, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108507

RESUMO

Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.


Assuntos
Comunicação Celular , Técnicas de Cocultura/métodos , Derme/metabolismo , Endotélio Vascular/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Pericitos/metabolismo , Retina/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Derme/citologia , Endotélio Vascular/citologia , Humanos , Pericitos/citologia , Retina/citologia
11.
J Virol ; 95(18): e0082121, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34191581

RESUMO

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. Distinct from other viruses studied to date, at mid- to late stages of its unusually protracted replication cycle, human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure, termed the assembly compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of transforming acidic coiled coil protein 3 (TACC3) to recruit the MT polymerase, chTOG, from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. IMPORTANCE Diverse viruses rely on host cell microtubule networks to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. However, precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the assembly compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid- to late stages of infection, HCMV increases the expression of transforming acidic coiled coil protein 3 (TACC3) to control the localization of the microtubule polymerase, chTOG. This, in turn, enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Fibroblastos/virologia , Complexo de Golgi/virologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Replicação Viral , Células Cultivadas , Derme/metabolismo , Derme/virologia , Fibroblastos/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/virologia
12.
FASEB J ; 35(6): e21627, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33948992

RESUMO

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.


Assuntos
Derme/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Junções Íntimas/fisiologia , Proteína rhoB de Ligação ao GTP/metabolismo , Permeabilidade Capilar , Derme/citologia , Derme/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteína rhoB de Ligação ao GTP/genética
13.
Theranostics ; 11(13): 6461-6476, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995668

RESUMO

Vascular endothelial cells (ECs) are increasingly recognized as active players in intercellular crosstalk more than passive linings of a conduit for nutrition delivery. Yet, their functional roles and heterogeneity in skin remain uncharacterized. We have used single-cell RNA sequencing (scRNA-seq) as a profiling strategy to investigate the tissue-specific features and intra-tissue heterogeneity in dermal ECs at single-cell level. Methods: Skin tissues collected from 10 donors were subjected to scRNA-seq. Human dermal EC atlas of over 23,000 single-cell transcriptomes was obtained and further analyzed. Arteriovenous markers discovered in scRNA-seq were validated in human skin samples via immunofluorescence. To illustrate tissue-specific characteristics of dermal ECs, ECs from other human tissues were extracted from previously reported data and compared with our transcriptomic data. Results: In comparison with ECs from other human tissues, dermal ECs possess unique characteristics in metabolism, cytokine signaling, chemotaxis, and cell adhesions. Within dermal ECs, 5 major subtypes were identified, which varied in molecular signatures and biological activities. Metabolic transcriptome analysis revealed a preference for oxidative phosphorylation in arteriole ECs when compared to capillary and venule ECs. Capillary ECs abundantly expressed HLA-II molecules, suggesting its immune-surveillance role. Post-capillary venule ECs, with high levels of adhesion molecules, were equipped with the capacity in immune cell arrest, adhesion, and infiltration. Conclusion: Our study provides a comprehensive characterization of EC features and heterogeneity in human dermis and sets the stage for future research in identifying disease-specific alterations of dermal ECs in various dermatoses.


Assuntos
Derme/citologia , Células Endoteliais/metabolismo , Transcriptoma , Sequência de Bases , Biomarcadores , Capilares/citologia , Adesão Celular , Derme/irrigação sanguínea , Derme/metabolismo , Expressão Gênica , Humanos , Fenótipo , Análise de Célula Única , Vênulas/citologia
14.
Mol Cell Biochem ; 476(10): 3613-3622, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34028646

RESUMO

Molecular hydrogen (H2) is recognized as a gaseous antioxidant, and it is expected to ameliorate various disorders related to oxidative stress and inflammation. However, there are still many unclear points regarding its effectiveness in the skin. Therefore, the purpose of this study was to examine the protective effect of H2 against ultraviolet (UV) irradiation-related stress injury in human epidermal HaCaT cells. We investigated the effects of H2 against three types of UV-derived oxidative stress using human skin keratinocytes: hydrogen peroxide (H2O2)-induced oxidative stress, tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation stress, and glyoxal-induced carbonyl stress. Our results showed that H2 exerted cytoprotective effects against stress induced by H2O2, t-BuOOH, and glyoxal. Furthermore, our results also revealed that H2 suppressed H2O2-induced increases in intracellular peroxide and H2O2 levels, and suppressed the progression of lipid peroxidation. Taken together, our results demonstrate that H2 can exert protective effects against oxidative stress-, lipid peroxidation-, and carbonyl stress-induced cellular injuries in human keratinocytes, partly mediated via suppression of intracellular oxidative stress and peroxide generation. Therefore, H2 is expected to be utilized as an effective and attractive component in cosmetic formulations in the future.


Assuntos
Derme/lesões , Glioxal/toxicidade , Peróxido de Hidrogênio/toxicidade , Hidrogênio/farmacologia , Queratinócitos/metabolismo , Linhagem Celular , Derme/metabolismo , Derme/patologia , Humanos , Queratinócitos/patologia
15.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946465

RESUMO

Cortisol is an endogenous glucocorticoid (GC) and primary stress hormone that regulates a wide range of stress responses in humans. The adverse effects of cortisol on the skin have been extensively documented but the underlying mechanism of cortisol-induced signaling is still unclear. In the present study, we investigate the effect of cortisol on collagen type I expression and the effect of AP collagen peptides, collagen tripeptide-rich hydrolysates containing 3% glycine-proline- hydroxyproline (Gly-Pro-Hyp, GPH) from the fish skin, on the cortisol-mediated inhibition of collagen type I and the cortisol-induced signaling that regulates collagen type I production in human dermal fibroblasts (HDFs). We determine that cortisol downregulates the expression of collagen type I. AP collagen peptides or GC receptor (GR) inhibitors recover the cortisol-mediated inhibition of collagen type I and GR activation. AP collagen peptides or GR inhibitors also prevent the cortisol-dependent inhibition of transforming growth factor (TGF)-ß signaling. AP collagen peptides or GR inhibitors are effective in the prevention of collagen type I inhibition mediated by cortisol in senescent HDFs and reconstituted human skin models. Taken together, GR signaling might be responsible for the cortisol-mediated inhibition of TGF-ß. AP collagen peptides act as GR-mediated signaling blockers, preventing the cortisol-dependent inhibition of collagen type I. Therefore, AP collagen peptides have the potential to improve skin health.


Assuntos
Anti-Inflamatórios/farmacologia , Colágeno Tipo I/metabolismo , Fibroblastos/efeitos dos fármacos , Hidrocortisona/farmacologia , Oligopeptídeos/farmacologia , Animais , Linhagem Celular , Derme/citologia , Derme/efeitos dos fármacos , Derme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Peixes , Humanos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
17.
Exp Cell Res ; 404(1): 112618, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33965401

RESUMO

Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from -100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.


Assuntos
Alopecia/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Folículo Piloso/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Alopecia/tratamento farmacológico , Alopecia/genética , Derme/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fatores de Transcrição/metabolismo
18.
J Adv Res ; 30: 103-112, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34026290

RESUMO

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- ß1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.


Assuntos
Folículo Piloso/citologia , Folículo Piloso/metabolismo , Cabelo/fisiologia , Queratinócitos/metabolismo , Regeneração , Animais , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/metabolismo , Derme/metabolismo , Epitélio/metabolismo , Humanos , Camundongos , Camundongos Nus , Fenótipo , Pele/metabolismo , Esferoides Celulares/citologia
19.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33921970

RESUMO

Recently, a variety of safe and effective non-pharmacological methods have been introduced as new treatments of alopecia. Micro-current electrical stimulation (MCS) is one of them. It is generally known to facilitate cell proliferation and differentiation and promote cell migration and ATP synthesis. This study aimed to investigate the hair growth-promoting effect of MCS on human hair follicle-derived papilla cells (HFDPC) and a telogenic mice model. We examined changes in cell proliferation, migration, and cell cycle progression with MCS-applied HFDPC. The changes of expression of the cell cycle regulatory proteins, molecules related to the PI3K/AKT/mTOR/Fox01 pathway and Wnt/ß-catenin pathway were also examined by immunoblotting. Subsequently, we evaluated the various growth factors in developing hair follicles by RT-PCR in MCS-applied (MCS) mice model. From the results, the MCS-applied groups with specific levels showed effects on HFDPC proliferation and migration and promoted cell cycle progression and the expression of cell cycle-related proteins. Moreover, these levels significantly activated the Wnt/ß-catenin pathway and PI3K/AKT/mTOR/Fox01 pathway. Various growth factors in developing hair follicles, including Wnts, FGFs, IGF-1, and VEGF-B except for VEGF-A, significantly increased in MCS-applied mice. Our results may confirm that MCS has hair growth-promoting effect on HFDPC as well as telogenic mice model, suggesting a potential treatment strategy for alopecia.


Assuntos
Derme/citologia , Terapia por Estimulação Elétrica/instrumentação , Terapia por Estimulação Elétrica/métodos , Folículo Piloso/citologia , Cabelo/crescimento & desenvolvimento , Transdução de Sinais , Animais , Ciclo Celular , Movimento Celular , Proliferação de Células , Derme/metabolismo , Regulação da Expressão Gênica , Folículo Piloso/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Nat Commun ; 12(1): 2147, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846309

RESUMO

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).


Assuntos
Canal Anal/citologia , Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Genitália/citologia , HIV-1/fisiologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Monócitos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Forma Celular , Colagenases/metabolismo , Derme/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Membrana Mucosa/metabolismo , Fagócitos/metabolismo , Fenótipo , Receptores CCR5/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transcrição Genética
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