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1.
mSphere ; 4(4)2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391281

RESUMO

Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.


Assuntos
Diferenciação Celular , Proliferação de Células , Papillomavirus Humano 16/fisiologia , Queratinócitos/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação Viral , Sistemas CRISPR-Cas , Células Cultivadas , Deleção de Genes , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Queratinócitos/virologia , Tonsila Palatina/citologia , Tonsila Palatina/virologia
2.
Vet Dermatol ; 30(5): 365-e107, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31297916

RESUMO

BACKGROUND: Nestin, which was originally described as a neural crest stem cell marker, is known to be expressed in bulge follicle cells of human, canine and murine anagen hairs. However, the capacity of nestin-expressing cells to differentiate into the components of the hair follicle or the epidermis has been insufficiently investigated. HYPOTHESIS/OBJECTIVES: To determine whether nestin-expressing cells are capable of differentiating into keratinocytes. ANIMALS/MATERIALS: A double-transgenic mouse line Nes-Cre/CAG-CAT-EGFP, in which enhanced green fluorescent protein (EGFP) is expressed upon Cre-based recombination driven by the nestin promoter. METHODS AND MATERIALS: The tissue distribution of EGFP+ and nestin+ cells in the skin of the mouse line was analysed by immunofluorescence and immunohistochemical analyses. RESULTS: EGFP+ cells were recognized in the outer epithelial cell layers of anagen and telogen hair follicles, but rarely seen in the interfollicular epidermis. The EGFP+ cells in the outer layers of the hair follicles coexpressed keratin 14, a marker of the outer root sheath (ORS) keratinocytes, but not trichohyalin granules, an inner root sheath keratinocyte cell marker. Immunostaining for nestin failed to detect its expression in the majority of hair follicle epithelial cells, suggesting that the EGFP+ cells in the ORS were derived from nestin-expressing progenitor cells that had become further committed along the epithelial cell lineage, where nestin is no longer expressed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest that progenitor cells that differentiate into ORS keratinocytes are distinct from those for other hair follicle or epidermal components and provide implications for regenerative medicine and the molecular classification of hair follicle tumours.


Assuntos
Diferenciação Celular/fisiologia , Folículo Piloso/citologia , Queratinócitos/classificação , Nestina/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Nestina/genética
3.
Vet Dermatol ; 30(5): 417-e126, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31328349

RESUMO

BACKGROUND: The re-epithelialization process in equine wound healing is incompletely described. For epithelial cells to migrate during embryogenesis they undergo epithelial-to-mesenchymal transition (EMT); this phenotypic transition occurs during wound healing in humans and rodents, but it has not been investigated in horses. HYPOTHESIS/OBJECTIVES: To investigate keratinocyte differentiation and EMT in equine experimental excisional limb and body wounds healing by second intention. ANIMALS: Six adult research horses. METHODS AND MATERIALS: Immunohistochemical analysis was used to detect expression of the differentiation markers cytokeratin (CK)10, CK14, loricrin and peroxisome proliferator-activated receptor alpha (PPAR-α), and of the EMT markers E-cadherin and N-cadherin in normal limb and body skin, and biopsies from limb and body wounds. RESULTS: Loricrin and CK10 were expressed in normal skin and periwound skin but not in migrating epithelium of body and limb wounds. However, they reappeared at the migrating epithelial tip of body wounds only. CK14 and PPAR-α had uniform distribution throughout the migrating epithelium. N-cadherin was not expressed in normal unwounded skin but was detected in periwound skin adjacent to the wound margin. E-cadherin expression decreased at the wound margin. CONCLUSIONS AND CLINICAL IMPORTANCE: Presence of N-cadherin suggests that cadherin switching occurred during wound healing, this may be an indication that EMT occurs in horses. To the best of the authors' knowledge, this has never been described in horses before and warrants further investigation to assess the clinical implications. The tip of the migrating epithelium in body wounds appeared more differentiated than limb wounds, which could be part of the explanation for the superior healing of body wounds.


Assuntos
Diferenciação Celular/fisiologia , Extremidades/lesões , Cavalos/lesões , Queratinócitos/fisiologia , Cicatrização/fisiologia , Animais , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Imuno-Histoquímica , Queratinas/genética , Queratinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo
4.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340560

RESUMO

BACKGROUND: Due to current antibiotic resistance worldwide, there is an urgent need to find new alternative antibacterial approaches capable of dealing with multidrug-resistant pathogens. Most recent studies have demonstrated the antibacterial activity and non-cytotoxicity of carbon nanomaterials such as graphene oxide (GO) and carbon nanofibers (CNFs). On the other hand, light-emitting diodes (LEDs) have shown great potential in a wide range of biomedical applications. METHODS: We investigated a nanotechnological strategy consisting of GO or CNFs combined with light-emitting diod (LED) irradiation as novel nanoweapons against two clinically relevant Gram-positive multidrug-resistant pathogens: methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). The cytotoxicity of GO and CNFs was studied in the presence of human keratinocyte HaCaT cells. RESULTS: GO or CNFs exhibited no cytotoxicity and high antibacterial activity in direct contact with MRSE and MRSA cells. Furthermore, when GO or CNFs were illuminated with LED light, the MRSE and MRSA cells lost viability. The rate of decrease in colony forming units from 0 to 3 h, measured per mL, increased to 98.5 ± 1.6% and 95.8 ± 1.4% for GO and 99.5 ± 0.6% and 99.7 ± 0.2% for CNFs. CONCLUSIONS: This combined antimicrobial approach opens up many biomedical research opportunities and provides an enhanced strategy for the prevention and treatment of Gram-positive multidrug-resistant infections.


Assuntos
Antibacterianos/farmacologia , Grafite/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos da radiação , Nanoestruturas/química , Nanotubos de Carbono/química , Staphylococcus epidermidis/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Técnicas de Cocultura , Relação Dose-Resposta à Radiação , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Queratinócitos/efeitos da radiação , Luz , Resistência a Meticilina/efeitos da radiação , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos da radiação , Nanoestruturas/ultraestrutura , Nanotubos de Carbono/ultraestrutura , Fototerapia/métodos , Staphylococcus epidermidis/crescimento & desenvolvimento
5.
PLoS One ; 14(7): e0219938, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31335913

RESUMO

Podoplanin (PDPN) is a glycoprotein that is expressed by various cell types, including keratinocytes, fibroblasts, and lymphatic endothelial cells. We found that PDPN is expressed in the hair follicle (HF) keratinocyte region and HF stem cell area during the late anagen phase but not during the telogen phase in mice. Importantly, keratinocyte-specific PDPN deletion in mice (K5-Cre;PDPNflox/flox) promoted anagen HF growth after depilation-induced HF regeneration as compared to control mice. RNA sequencing, followed by gene ontology analysis, showed down-regulation of focal adhesion and extracellular matrix interaction pathways in HF stem cells isolated from K5-Cre;PDPNflox/flox mice as compared to control mice. Furthermore, HF keratinocytes isolated from K5-Cre;PDPNflox/flox mice exhibited a decreased ability to interact with collagen type I in cell adhesion assays. Taken together, these results show that PDPN deletion promotes HF cycling, possibly via reduced focal adhesion and concomitantly enhanced migration of HF stem cells towards the bulb region. They also indicate potential new therapeutic strategies for the treatment of conditions associated with hair loss.


Assuntos
Folículo Piloso/crescimento & desenvolvimento , Glicoproteínas de Membrana/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Animais , Movimento Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL
6.
Iran J Immunol ; 16(2): 97-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182684

RESUMO

Atopic dermatitis (AD) is characterized by skin inflammation, barrier dysfunction and chronic pruritus. In this review, recent advances in the pathogenesis of AD are summarized. Clinical efficacy of the anti-IL-4 receptor antibody dupilumab implies that type 2 cytokines IL-4 and IL-13 have pivotal roles in atopic inflammation. The expression of IL-4 and IL-13 as well as type 2 chemokines such as CCL17, CCL22 and CCL26 is increased in the lesional skin of AD. In addition, IL-4 and IL-13 down-regulate the expression of filaggrin in keratinocytes and exacerbate epidermal barrier dysfunction. Keratinocytes in barrier-disrupted epidermis produce large amounts of thymic stromal lymphopoietin, IL-25 and IL-33, conducing to type 2 immune deviation via OX40L/OX40 signaling. IL-31, produced by type 2 T cells, is a cardinal pruritogenic cytokine. IL-4 and IL-13 also amplify the IL-31-mediated sensory nerve signal. These molecules are particularly important targets for future drug development for AD.


Assuntos
Dermatite Atópica/imunologia , Epiderme/patologia , Queratinócitos/fisiologia , Células Th2/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Citocinas/metabolismo , Dermatite Atópica/tratamento farmacológico , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Ligante OX40/metabolismo , Receptores de Interleucina-4/imunologia , Receptores OX40/metabolismo , Transdução de Sinais
7.
Methods Mol Biol ; 1993: 181-190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148087

RESUMO

Isolation and culture of keratinocytes from patients with different types of epidermolysis bullosa are sometimes challenging, because of the inherent adhesion defects of these cells. We routinely employ a well-established protocol for in vitro culture of these cells from small skin samples remaining after diagnostic procedures are performed. Keratinocytes and fibroblast can be used for downstream expression and functional studies or for construction of in vitro organotypic cocultures. These cells maintain main common characteristics of spreading, adhesion, migration, and survival, which depend on the underlying molecular defect.


Assuntos
Técnicas de Cocultura/métodos , Epidermólise Bolhosa/fisiopatologia , Fibroblastos/fisiologia , Queratinócitos/fisiologia , Adesão Celular , Movimento Celular , Separação Celular , Sobrevivência Celular , Humanos , Pele/citologia
8.
Arch Dermatol Res ; 311(8): 647-652, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31168656

RESUMO

In this study, we characterised the effect that seeding keratinocytes on the papillary and reticular dermis had on the extracellular matrix and tissue integrity ex vivo. Human skin explants from consented patients (n = 6) undergoing routine surgery were cultured at a liquid-air interface, dermal-side up, and autologous keratinocytes seeded on the exposed papillary or reticular layer. After 7-21 days, histological and immunohistochemical evaluation of the morphology and extracellular matrix was performed. While the dermis remained robust in all explants cultures, keratinocytes seeded on the papillary layer showed less tissue infiltration and remodelling and formed clusters across the tissue. In contrast, keratinocytes seeded on the reticular layer infiltrated the tissue homogenously with an intact single-cell-layer surface coverage and structural changes characterised by increased deposition of ground substance, glycosaminoglycans, and collagen VII in 14 days. In addition, while the papillary section showed more new laminin deposition by 14 days than the reticular section, the latter expressed more connexin 43. These differences in re-epithelialisation and extracellular matrix characteristics suggest that wound depth and graft thickness may play a key role in wound healing and indicate that ECM characteristics should be factored in when designing biomaterials for wound applications and in the selection of recipient sites when using cells for grafting.


Assuntos
Matriz Extracelular/metabolismo , Queratinócitos/fisiologia , Reepitelização/fisiologia , Adulto , Idoso , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Pessoa de Meia-Idade , Pele/citologia , Pele/metabolismo , Ferimentos e Lesões/terapia
9.
Life Sci ; 230: 28-34, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31108094

RESUMO

Psoriasis, a chronic inflammatory skin disorder disease, is closely associated with hyperproliferation of keratinocytes. Upregulated miR-223 has been found in peripheral blood mononuclear cells from patients with psoriasis and from psoriatic skin. However, its role in keratinocytes remains unknown. We thus aimed to investigate the function of miR-223 in psoriasis. Interleukin-22 (IL-22) is a crucial keratinocyte trigger in the T-cell-mediated immune response to psoriasis. We found miR-223 to be overexpressed in psoriatic lesions and in IL-22-stimulated HaCaT cells. HaCaT cells then were transfected with a miR-223 mimic or inhibitor to overexpress or inhibit expression of miR-223, respectively. A Cell Counting Kit-8 assay revealed that miR-223 overexpression promoted and miR-223 downregulation inhibited proliferation in IL-22-stimulated HaCaT cells. Flow cytometry analysis certified that miR-223 overexpression decreased HaCaT cell apoptosis, whereas miR-223 downregulation increased it. A dual-luciferase reporter assay demonstrated that miR-223 directly targeted the phosphatase and tensin homolog (PTEN) gene. MiR-223 also negatively regulated mRNA and protein expression of PTEN and modulated the PTEN/Akt pathway in IL-22-stimulated HaCaT cells. PTEN silencing attenuated the activity of the miR-223 inhibitor in these cells via the PTEN/Akt pathway. Overall, the results showed that miR-223 increased proliferation and inhibited apoptosis of IL-22-stimulated keratinocytes via the PTEN/Akt pathway.


Assuntos
Queratinócitos/fisiologia , MicroRNAs/fisiologia , Psoríase/genética , Apoptose/genética , Linhagem Celular , Proliferação de Células/genética , Humanos , Interleucinas/genética , Interleucinas/imunologia , Queratinócitos/metabolismo , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética
10.
Int Immunopharmacol ; 72: 511-521, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075711

RESUMO

Shikonin is an active compound of the oriental medicinal plant, Leptospermum erythrorhizon, which has been previously shown to inhibit psoriasis-like inflammation. However, the underlying mechanism is unclear. In the present study, the mechanisms of keratinocyte proliferation and apoptosis in psoriasis in response to shikonin were explored both in vitro and in vivo. Our results showed that shikonin significantly inhibits cell proliferation and induces apoptosis in both HaCaT and LV-STAT3 HaCaT cells by targeting CEBPD, while a decrease in cell survival, proliferation and viability were found through flow-cytometry and MTS assay. Furthermore, gavage with shikonin markedly alleviated psoriasis-like manifestations in IMQ-induced BALB/c mice clinically (PASI Score) and histopathologically. Immunohistochemistry revealed that shikonin potently suppresses the JAK/STAT3 signaling pathway in local skin lesions and increases CEBPD expression. These results imply that shikonin inhibits keratinocyte proliferation and induces apoptosis, which results in psoriasis treatment through the JAK/STAT3 dependent pathway. In addition, the activation of JAK/STAT3 downregulates CEBPD in HaCaT cells and IMQ-induced BALB/c mice. However, shikonin can reverse these effects, suggesting that CEBPD may be a potential therapeutic target for psoriasis.


Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Queratinócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Imiquimode , Janus Quinases , Queratinócitos/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Regulação para Cima/efeitos dos fármacos
11.
Int J Mol Sci ; 20(10)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137673

RESUMO

Natural products or herbs can be used as an effective therapy for treating psoriasis, an autoimmune skin disease that involves keratinocyte overproliferation. It has been demonstrated that phytomedicine, which is used for psoriasis patients, provides some advantages, including natural sources, a lower risk of adverse effects, and the avoidance of dissatisfaction with conventional therapy. The herbal products' structural diversity and multiple mechanisms of action have enabled the synergistic activity to mitigate psoriasis. In recent years, the concept of using natural products as antiproliferative agents in psoriasis treatment has attracted increasing attention in basic and clinical investigations. This review highlights the development of an apoptotic or antiproliferatic strategy for natural-product management in the treatment of psoriasis. We systematically introduce the concepts and molecular mechanisms of keratinocyte-proliferation inhibition by crude extracts or natural compounds that were isolated from natural resources, especially plants. Most of these studies focus on evaluation through an in vitro keratinocyte model and an in vivo psoriasis-like animal model. Topical delivery is the major route for the in vivo or clinical administration of these natural products. The potential use of antiproliferative phytomedicine on hyperproliferative keratinocytes suggests a way forward for generating advances in the field of psoriasis therapy.


Assuntos
Produtos Biológicos/uso terapêutico , Queratinócitos/efeitos dos fármacos , Psoríase/tratamento farmacológico , Animais , Apoptose , Produtos Biológicos/farmacologia , Proliferação de Células , Humanos , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Psoríase/metabolismo
12.
Int J Radiat Oncol Biol Phys ; 105(1): 193-205, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31085283

RESUMO

PURPOSE: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). METHODS AND MATERIALS: Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. RESULTS: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs. CONCLUSIONS: We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.


Assuntos
Senescência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Células-Tronco Pluripotentes Induzidas/citologia , Queratinócitos/efeitos da radiação , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores/análise , Diferenciação Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Raios gama , Histonas/análise , Humanos , Queratina-14/análise , Queratinócitos/química , Queratinócitos/fisiologia , Proteínas de Membrana/análise , Receptores da Transferrina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/análise
14.
Toxicol In Vitro ; 60: 27-35, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31028861

RESUMO

The molecular pathways by which flavagline derivatives exert their cytotoxicity against various cancer cell types are well documented, while the mechanisms that prevent their cytotoxic effects on normal cells still have to be clarified. Here we provide the underlying molecular events by which normal skin cells remain unaffected after exposure to the synthetic flavagline FL3. Indeed, the anticancer agent fails to trigger apoptosis of healthy cells and is unable to induce the depolarization of their mitochondrial membrane and the cytosolic release of cytochrome C, in contrast to what is observed for cancer cells. Most importantly, FL3 specifically induces in normal cells, but not in malignant cells, an activation of Bad, without significant mitochondrial and cytosolic redistribution of Bax or Bcl-2. Moreover, gene knockdown of Bad sensitizes the normal fibroblastic cells to FL3 and induces a caspase-3 dependent apoptosis. Bad activation, known to promote survival and block apoptosis, explains therefore the lack of cytotoxicity of FL3 on normal skin cells. Finally, these findings provide new insights into the molecular mechanisms of resistance of healthy cells against FL3 cytotoxicity and identify it as a promising anticancer drug.


Assuntos
Antineoplásicos/farmacologia , Pele/citologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo
15.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970577

RESUMO

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of the epidermal cells and is clinically presented as thick, bright red to pink plaques with a silvery scale. Photodynamic therapy (PDT) using visible light has become of increasing interest in the treatment of inflammatory skin diseases. In this study, we demonstrate that a combination of curcumin-loaded chitosan/alginate nanoparticles (Cur-CS/Alg NPs) and blue light emitting diodes (LED) light irradiation effectively suppressed the hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced cultured human kerlatinocyte (HaCaT) cells. The Cur-CS/Alg NPs were fabricated by emulsification of curcumin in aqueous sodium alginate solution and ionotropic gelation with calcium chloride and chitosan using an optimized formulation derived from a Box-Behnken design. The fabricated Cur-CS/Alg NPs were characterized for their particle size, zeta potential, encapsulation efficiency, and loading capacity. The surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, to measure the relative number of viable cells, showed that the CS/Alg NPs were nontoxic to normal HaCaT cells, while 0.05 µg/mL and 0.1 µg/mL of free curcumin and Cur-CS/Alg NPs inhibited the hyperproliferation of HaCaT cells induced by TNF-α. However, the Cur-CS/Alg NPs demonstrated a stronger effect than the free curcumin, especially when combined with blue light irradiation (10 J/cm²) from an LED-based illumination device. Therefore, the Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis.


Assuntos
Proliferação de Células , Sistemas de Liberação de Medicamentos , Queratinócitos/metabolismo , Luz , Nanopartículas/química , Psoríase/terapia , Fator de Necrose Tumoral alfa/farmacologia , Alginatos/química , Alginatos/farmacologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quitosana/química , Quitosana/farmacologia , Curcumina/química , Curcumina/farmacologia , Humanos , Queratinócitos/fisiologia , Psoríase/metabolismo , Psoríase/patologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Skin Pharmacol Physiol ; 32(3): 162-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31018213

RESUMO

BACKGROUND: The number of patients who has a daily intake of antihypertensive drugs is rising, due to an also rising prevalence of lifestyle diseases. Interestingly, knowledge about effects of these drugs in terms of wound healing is low. OBJECTIVE: Based on a few differing studies, the idea arose that antihypertensives may have side effects on wound healing. METHODS: Five antihypertensive drugs from different substance classes (metoprolol, amlodipine, ramipril, hydrochlorothiazide, candesartan) were investigated, in terms of possible impacts on cell metabolism and migration of human skin fibroblasts and keratinocytes. Additionally, histological and immunohistochemical analyses were performed in a 3-dimensional (3D) wound model addressing the influence on regeneration processes, such as cell migration, metabolic activity, apoptosis and epidermal thickness. RESULTS: Hydrochlorothiazide and ramipril exerted inhibiting effects in nearly all analyses, interestingly, in serum equivalent doses. In contrast, candesartan and amlodipine induced slight positive effects in 2D as well as in 3D models. The previously described positive effects of ß-blockers could only partially be confirmed by metoprolol. Antihypertensive drugs affected fibroblasts more than keratinocytes - whether positively or negatively. CONCLUSION: Antihypertensive drugs have an influence on keratinocytes and fibroblasts; they are not neutral. Candesartan has the most positive effects on skin cells. For angiotensin-converting enzyme inhibitors and thiazide diuretics, wound healing in a 3D model is delayed. ß-Receptor blockers seem to improve wound healing to a small extent just like calcium channel blockers. These results should be evaluated in a clinical trial to verify their clinical relevance.


Assuntos
Anti-Hipertensivos/farmacologia , Fibroblastos/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Anlodipino/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Benzimidazóis/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Diuréticos/farmacologia , Fibroblastos/fisiologia , Humanos , Hidroclorotiazida/farmacologia , Queratinócitos/fisiologia , Metoprolol/farmacologia , Ramipril/farmacologia , Tetrazóis/farmacologia
18.
J Ethnopharmacol ; 238: 111844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-30940568

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Several traditional medicinal herbs are widely used for dermatologic and cosmetic preparations. The beneficial skin repair activity is detected in various phases of wound-healing process, such as cell-cell, cell-matrix interactions or collagen synthesis. AIM OF THE STUDY: The study assessed the effects of Opuntia ficus-indica (L.) Mill. (Opuntia) and Milk Thistle (MT) (Silybum marianum (L.) Gaerth) on adult keratinocytes (HaCaT) functioning under basal condition or in the presence of mechanical damage (wounded cells). MATERIALS AND METHODS: The role of the natural compounds was tested on HaCaT grown in mono-culture and tri-culture configurations. In tri-cultures models, HaCaT were treated with the conditioned media (CM) obtained by Human Normal Dermal Fibroblast (NHDF) and Human Dermal Microvascular Endothelial cells (HMVEC) co-cultures. Specifically, were tested cell viability, oxidative stress mechanisms (cytokines release and lipid peroxidation) and cellular remodelling (growth factors release or metalloproteinase modulation). Moreover, the migratory potential of HaCaT was analysed by the use of wound healing in vitro assay. RESULTS: Opuntia and MT differently modified the metabolism (EGF, MMP-9), and the migratory properties of HaCaT both under physiological conditions or upon mechanical damage (wounded cells). Moreover, both compounds modulated HaCaT response to oxidative stress. The response to the natural compounds were modified, and in some cases potentiated, in tri-culture configuration systems. CONCLUSIONS: The data demonstrated that in vitro tri-culture approach is suitable to characterize the role of natural compounds on the complex communication between dermal-epidermal cellular components and microvascular endothelium. Specifically, Opuntia and MT are good alternatives to synthetic compounds in skin repair promotion.


Assuntos
Antioxidantes/farmacologia , Células Endoteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Flavonoides/farmacologia , Queratinócitos/efeitos dos fármacos , Cardo Mariano , Opuntia , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Dieta , Células Endoteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Queratinócitos/fisiologia , Cicatrização/efeitos dos fármacos
19.
Int Arch Allergy Immunol ; 179(1): 53-61, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30909282

RESUMO

BACKGROUND: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitary-adrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. METHODS: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. RESULTS: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. CONCLUSION: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression.


Assuntos
Dexametasona/farmacologia , Fator 7 de Crescimento de Fibroblastos/análise , Queratinócitos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Queratinócitos/fisiologia , Mastócitos/química , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C
20.
Arch Dermatol Res ; 311(4): 317-324, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847563

RESUMO

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery.


Assuntos
Permeabilidade da Membrana Celular , Epiderme/fisiologia , Frutose/metabolismo , Queratinócitos/fisiologia , Bicamadas Lipídicas/metabolismo , Xilitol/metabolismo , Adulto , Células Cultivadas , Exocitose , Homeostase , Humanos , Lipídeos/análise , Masculino , Fluidez de Membrana , Modelos Biológicos , Análise de Célula Única , Adulto Jovem
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