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1.
Braz. j. oral sci ; 20: e210053, jan.-dez. 2021. tab
Artigo em Inglês | LILACS, BBO - Odontologia | ID: biblio-1253163

RESUMO

Aim: The literature has not yet reported investigations about the effect of laser photobiomodulation (LPBM) over the cytotoxicity of drugs for endodontic treatments. Thus, the aim of this study was to evaluate, in vitro, the effect of the association between LPBM and intracanal medications on fibroblasts viability in different exposure times. Methods: Calcium hydroxide (Ca(OH)2) and iodoform (IO) were used pure or associated to LPBM. Eluates of medications were prepared and placed in contact with the cells in three different periods: 24h, 48h and 72h. Laser irradiation (emitting radiation λ 660nm, power density of 10mW, energy density of 3 J/cm²) has been performed in two sessions within a six hour interval, for 12s per well. After each experimental time, the colorimetric assay (MTT) has been performed. Statistical analysis was applied for Mann-Whitney test with 5% α error admitted test. Results: At 24h, the use of LPBM did not increase cell viability while after 72h cell proliferation was stimulated in the group without medications. LPBM application did not increase cell viability in Ca(OH)2 group and IO at any tested time. Ca(OH)2 cytotoxicity at 24h was higher than iodoform, while at 72h not difference was observed. Therefore, after 72 hours was no statistical difference between the IO and Ca(OH)2 groups. Conclusion: LPBM was able to increase cell viability in 72h in the group without medication, although no improvement was observed in the other groups. Thus, LPBM was not able to reduce the cytotoxic effects of the materials on fibroblasts in vitro


Assuntos
Terapia com Luz de Baixa Intensidade , Endodontia , Fibroblastos
2.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198528

RESUMO

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 µM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.


Assuntos
Proteínas de Transporte de Cátions/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Sinoviócitos/metabolismo , Canal de Cátion TRPA1/genética , Regulação para Cima/genética , Zinco/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/patologia , Espaço Intracelular/metabolismo , Sinoviócitos/patologia , Canal de Cátion TRPA1/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198681

RESUMO

Lack of adult cells' ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.


Assuntos
Monofosfato de Adenosina/farmacologia , Derme/fisiologia , Elastina/biossíntese , Estradiol/farmacologia , Caracteres Sexuais , Pele Artificial , Engenharia Tecidual , Adulto , Meios de Cultura , AMP Cíclico/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto Jovem
4.
Molecules ; 26(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201111

RESUMO

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA5m.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [68Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA5 chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA5.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA5.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC50 values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA5.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA5.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.


Assuntos
Acetatos/farmacologia , Azepinas/farmacologia , Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos com 1 Anel/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Quinina/análogos & derivados , Endopeptidases , Fibroblastos/metabolismo , Radioisótopos de Gálio/farmacologia , Humanos , Ligantes , Lutécio/farmacologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Quinina/farmacologia , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Escândio/farmacologia , Serina Endopeptidases/metabolismo
5.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204949

RESUMO

Idiopathic pulmonary fibrosis (IPF) is one of the most symptomatic progressive fibrotic lung diseases, in which patients have an extremely poor prognosis. Therefore, understanding the precise molecular mechanisms underlying pulmonary fibrosis is necessary for the development of new therapeutic options. Stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) are ubiquitously expressed in various types of cells and activated in response to cellular environmental stresses, including inflammatory and apoptotic stimuli. Type II alveolar epithelial cells, fibroblasts, and macrophages are known to participate in the progression of pulmonary fibrosis. SAPKs can control fibrogenesis by regulating the cellular processes and molecular functions in various types of lung cells (including cells of the epithelium, interstitial connective tissue, blood vessels, and hematopoietic and lymphoid tissue), all aspects of which remain to be elucidated. We recently reported that the stepwise elevation of intrinsic p38 signaling in the lungs is correlated with a worsening severity of bleomycin-induced fibrosis, indicating an importance of this pathway in the progression of pulmonary fibrosis. In addition, a transcriptome analysis of RNA-sequencing data from this unique model demonstrated that several lines of mechanisms are involved in the pathogenesis of pulmonary fibrosis, which provides a basis for further studies. Here, we review the accumulating evidence for the spatial and temporal roles of SAPKs in pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase 4/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/crescimento & desenvolvimento , Fibroblastos/enzimologia , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/patologia , Pulmão/embriologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/enzimologia
6.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199374

RESUMO

BACKGROUND: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. METHODS: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. RESULTS: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels' ability to prompt elastin expression and in ColIV/ColI ratio. CONCLUSIONS: A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels' behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.


Assuntos
Ácido Hialurônico/química , Hialuronoglucosaminidase/genética , Hidrogéis/química , Pele/efeitos dos fármacos , Elastina/química , Fibroblastos/efeitos dos fármacos , Humanos , Hialuronoglucosaminidase/química , Injeções , Espécies Reativas de Oxigênio/química , Rejuvenescimento/fisiologia , Reologia , Pele/crescimento & desenvolvimento , Pele/patologia , Envelhecimento da Pele/genética , Viscosidade
7.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204432

RESUMO

Several transmembrane mucins have demonstrated that they contribute intracellularly to induce fibrotic processes. The extracellular domain of MUC16 is considered as a biomarker for disease progression and death in IPF patients. However, there is no evidence regarding the signalling capabilities of MUC16 that contribute to IPF development. Here, we demonstrate that MUC16 was overexpressed in the lung tissue of IPF patients (n = 20) compared with healthy subjects (n = 17) and localised in fibroblasts and hyperplastic alveolar type II cells. Repression of MUC16 expression by siRNA-MUC16 transfection inhibited the TGF-ß1-induced fibrotic processes such as mesenchymal/ myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as fibroblast proliferation. SiRNA-MUC16 transfection also decreased the TGF-ß1-induced SMAD3 phosphorylation, thus inhibiting the Smad Binding Element activation. Immunoprecipitation assays and confocal immunofluorescence showed the formation of a protein complex between MUC16/p-SMAD3 in the cell membrane after TGF-ß1 stimulation. This study shows that MUC16 is overexpressed in IPF and collaborates with the TGF-ß1 canonical pathway to induce fibrotic processes. Therefore, direct or indirect targeting of MUC16 could be a potential drug target for human IPF.


Assuntos
Antígeno Ca-125/genética , Expressão Gênica , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Idoso , Biomarcadores , Antígeno Ca-125/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Miofibroblastos/metabolismo , Fosforilação , Testes de Função Respiratória
8.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199748

RESUMO

BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.


Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Psoríase/genética , Adulto , Idoso , Biópsia , Conexinas/metabolismo , Conexinas/farmacologia , Epiderme/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Mediadores da Inflamação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Modelos Biológicos , Oligopeptídeos/farmacologia , Peptidoglicano/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Psoríase/patologia , Staphylococcus aureus/fisiologia , Adulto Jovem
9.
Anticancer Res ; 41(7): 3293-3298, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230124

RESUMO

BACKGROUND/AIM: Plexiform neurofibromas (PNFs) are benign tumors composed mainly of tumorous Schwann cells and non-tumorous fibroblasts. This study examined the possible enhancing effect of vitamin D on the efficacy of drugs used for the treatment of PNF in vitro. MATERIALS AND METHODS: Paired Schwann cells and fibroblasts were cultured from 10 PNFs and treated with imatinib and nilotinib in the absence and presence of calcipotriol, an analogue of the active metabolite of vitamin D. IC50 values for cell proliferation were calculated. RESULTS: Calcipotriol reduced the IC50 of the two drugs in both tumorous Schwann cells and non-tumorous fibroblasts by 40 to 45%. CONCLUSION: Calcipotriol enhanced the efficacy of imatinib and nilotinib on PNF-derived cells in vitro, though rather non-specifically. Nevertheless, sustaining vitamin D at 100-200 nM, the physiological range, may be beneficial for reducing the dose of drugs without scarifying efficacy.


Assuntos
Calcitriol/análogos & derivados , Mesilato de Imatinib/farmacologia , Neurofibroma Plexiforme/tratamento farmacológico , Pirimidinas/farmacologia , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Células de Schwann/efeitos dos fármacos , Vitamina D/farmacologia
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(8): 687-692, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34236028

RESUMO

Objective To investigate the effect of N-acetylcysteine (NAC) on the proliferation of fibroblast-like synoviocytes (FLS) treated with low concentration of hydrogen peroxide (H2O2) in rats with adjuvant arthritis (AA) and its mechanism. Methods Twenty SD rats were divided into a normal group and a model group (10 rats in each group). The model group was established by subcutaneous injection of Freund's complete adjuvant into the toe of rats, and the rats were sacrificed 28 days later. The contents of serum malondialdehyde (MDA) were detected by thiobarbituric acid method; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by hydroxylamine method and colorimetry respectively; and Nrf2 and Keap1 proteins in ankle synovial tissues of AA rats were detected by immunohistochemistry. AA-FLS were isolated, cultured, and identified by digestion of ankle joint slides of AA rats in vitro. The effects of NAC at different concentrations (final concentration 0, 0.3, 0.9, 3, 10, 30, 90, 180 µmol/L) on the activity of AA-FLS treated with H2O2 at low concentration (5 µmol/L) were detected by CCK-8 assay. The content of mitochondrial reactive oxygen species (ROS) in AA-FLS was detected by MitoSOX fluorescent probe. The effects of NAC (final concentration 0, 3, 10, 30 µmol/L) on Nrf2 and Keap1 protein expressions in AA-FLS treated with H2O2 at low concentration were detected by Western blotting. Results Compared with those in the control group, in AA model, the MDA level increased and SOD and GSH-Px levels decreased in serum, and the Nrf2 protein increased and the Keap1 protein decreased in synovial tissue. Immunocytochemical staining confirmed that the isolated and cultured cells were AA-FLS; NAC inhibited the proliferation of AA-FLS treated with H2O2 in a concentration-dependent manner, and the mitochondrial ROS content and the protein expressions of Nrf2 and Keap1 decreased. Conclusion NAC can inhibit the proliferation of AA-FLS treated with H2O2, which may be related to blocking Nrf2/Keap1 pathway.


Assuntos
Artrite Experimental , Sinoviócitos , Acetilcisteína/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Proliferação de Células , Fibroblastos/metabolismo , Peróxido de Hidrogênio , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Sprague-Dawley , Sinoviócitos/metabolismo
11.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208590

RESUMO

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.


Assuntos
Condrócitos/metabolismo , Fibroblastos/metabolismo , Osteoartrite/metabolismo , Proteoma , Proteômica , Membrana Sinovial/citologia , Peptídeo Intestinal Vasoativo/metabolismo , Biomarcadores , Condrócitos/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Suscetibilidade a Doenças , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Osteoartrite/etiologia , Osteoartrite/patologia , Proteômica/métodos , Peptídeo Intestinal Vasoativo/farmacologia
12.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208633

RESUMO

The clinical application of human platelet lysate (HPL) holds promise for tissue regeneration, and the development of an efficient vehicle for its delivery is desired. Chitosan-based hydrogels are potential candidates, but they often exhibit weak mechanical properties. In this study, a chitosan/gelatin (CS-GE) hydrogel crosslinked by glyoxal was fabricated for sustained release of HPL. The influence of HPL on Hs68 fibroblast and human umbilical vein endothelial cell (HUVEC) culture was evaluated, and we found that supplementing 5% HPL in the medium could significantly improve cell proliferation relative to supplementing 10% fetal bovine serum (FBS). Moreover, HPL accelerated the in vitro wound closure of Hs68 cells and facilitated the tube formation of HUVECs. Subsequently, we fabricated CS-GE hydrogels crosslinked with different concentrations of glyoxal, and the release pattern of FITC-dextrans (4, 40 and 500 kDa) from the hydrogels was assessed. After an ideal glyoxal concentration was determined, we further characterized the crosslinked CS-GE hydrogels encapsulated with different amounts of HPL. The HPL-incorporated hydrogel was shown to significantly promote the proliferation of Hs68 cells and the migration of HUVECs. Moreover, the release pattern of transforming growth factor-ß1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB) from hydrogel was examined in vitro, demonstrating a sustained release profile of the growth factors. Finally, the chick chorioallantoic membrane assay revealed that HPL encapsulation in the hydrogel significantly stimulated angiogenesis in ovo. These results demonstrate the great potential of the crosslinked CS-GE hydrogel to serve as an effective delivery system for HPL to promote tissue regeneration.


Assuntos
Produtos Biológicos/farmacologia , Plaquetas/metabolismo , Quitosana , Gelatina , Glioxal , Hidrogéis , Regeneração/efeitos dos fármacos , Proliferação de Células , Quitosana/química , Dextranos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatina/química , Glioxal/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/química , Neovascularização Fisiológica , Porosidade , Cicatrização/efeitos dos fármacos
13.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204292

RESUMO

Skin injury is quite common, and the wound healing is a complex process involving many types of cells, the extracellular matrix, and soluble mediators. Cell differentiation, migration, and proliferation are essential in restoring the integrity of the injured tissue. Despite the advances in science and technology, we have yet to find the ideal dressing that can support the healing of cutaneous wounds effectively, particularly for difficult-to-heal chronic wounds such as diabetic foot ulcers, bed sores, and venous ulcers. Hence, there is a need to identify and incorporate new ideas and methods to design a more effective dressing that not only can expedite wound healing but also can reduce scarring. Calcium has been identified to influence the wound healing process. This review explores the functions and roles of calcium in skin regeneration and reconstruction during would healing. Furthermore, this review also investigates the possibility of incorporating calcium into scaffolds and examines how it modulates cutaneous wound healing. In summary, the preliminary findings are promising. However, some challenges remain to be addressed before calcium can be used for cutaneous wound healing in clinical settings.


Assuntos
Cálcio/metabolismo , Cicatrização/fisiologia , Animais , Bandagens , Cálcio/farmacologia , Cálcio na Dieta/administração & dosagem , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Nanopartículas/química , Neovascularização Fisiológica , Regeneração , Pele/lesões , Pele/metabolismo , Nanomedicina Teranóstica , Engenharia Tecidual , Tecidos Suporte , Cicatrização/efeitos dos fármacos
14.
Nat Commun ; 12(1): 4229, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244477

RESUMO

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pinças Ópticas , Paxilina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Respiração , Organismos Livres de Patógenos Específicos , Talina/genética , Talina/metabolismo
15.
Arkh Patol ; 83(4): 22-28, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34278757

RESUMO

INTRODUCTION: Tumor budding was declared as independent prognostic factor for early cancer in 2016. Tumor-associated fibroblasts (CAFs) are one of the main components of tumor microenvironment. Plenty of different markers are used for detection of CAFs, including podoplanin (POD). OBJECTIVE: The aim of this study is to identify correlation between tumor budding, that indicates tumor invasive potential and is considered to be a negative prognostic factor, and CAFs near tumor buds using POD. MATERIAL AND METHODS: 43 cases of colon adenocarcinoma are included in the study. Double staining immunohistochemical technology with PCK and POD was used for detection of tumor budding and evaluation of POD expression near tumor buds. RESULTS: Significant correlations are revealed between tumor budding and depth of tumor invasion (p=0.023)/regional lymph node metastasis (p=0.068), but between POD expression and depth of tumor invasion (p=0.088) only a tendency of correlation is identified. These facts demonstrate critical prognostic value of tumor budding instead POD expression near tumor buds. It was found that intensity of POD expression near tumor buds is statistically analogous to POD expression in the invasive margin (p=0.0016). That means it is not necessary to evaluate POD expression exactly near tumor buds. For the first time stronger POD expression near mucin complexes is reported. That, probably, will allow to use mucin complexes as alternative prognostic factor instead tumor budding, as tumor buds are absent in mucinous adenocarcinoma.


Assuntos
Adenocarcinoma , Fibroblastos Associados a Câncer , Adenocarcinoma/genética , Biomarcadores Tumorais , Fibroblastos , Humanos , Metástase Linfática , Prognóstico , Microambiente Tumoral
16.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072418

RESUMO

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization-12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.


Assuntos
Biofilmes/crescimento & desenvolvimento , Técnicas de Cocultura , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Sobrevivência Celular , Fibroblastos/ultraestrutura , Imunofluorescência , Interações Hospedeiro-Patógeno , Cinética , Espectroscopia de Ressonância Magnética , Metaboloma , Metabolômica/métodos , Staphylococcus aureus/ultraestrutura
17.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072531

RESUMO

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.


Assuntos
Técnicas de Reprogramação Celular , Clonagem de Organismos , Embrião de Mamíferos , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Apoptose/genética , Biomarcadores , Bovinos , Clonagem de Organismos/métodos , Metilação de DNA , Implantação do Embrião , Epigênese Genética , Feminino , Fibroblastos , Expressão Gênica , Histonas/metabolismo , Gravidez , Sensibilidade e Especificidade , Doadores de Tecidos
18.
Int J Nanomedicine ; 16: 3707-3724, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34103912

RESUMO

Introduction: Intracellular delivery of molecules is central to applications in biotechnology, medicine, and basic research. Nanoparticle-mediated photoporation using carbon black nanoparticles exposed to pulsed, near-infrared laser irradiation offers a physical route to create transient cell membrane pores, enabling intracellular delivery. However, nanoparticle-mediated photoporation, like other physical intracellular delivery technologies, necessitates a trade-off between achieving efficient uptake of exogenous molecules and maintaining high cell viability. Methods: In this study, we sought to shift this balance by adding serum to cells during nanoparticle-mediated photoporation as a viability protectant. DU-145 prostate cancer cells and human dermal fibroblasts were exposed to laser irradiation in the presence of carbon black (CB) nanoparticles and other formulation additives, including fetal bovine serum (FBS) and polymers. Results: Our studies showed that FBS can protect cells from viability loss, even at high-fluence laser irradiation conditions that lead to high levels of intracellular delivery in two different mammalian cell types. Further studies revealed that full FBS was not needed: viability protection was achieved with denatured FBS, with just the high molecular weight fraction of FBS (>30 kDa), or even with individual proteins like albumin or hemoglobin. Finally, we found that viability protection was also obtained using certain neutral water-soluble polymers, including Pluronic F127, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), and polyethylene glycol, which were more effective at increased concentration, molecular weight, or hydrophobicity. Conclusion: Altogether, these findings suggest an interaction between amphiphilic domains of polymers with the cell membrane to help cells maintain viability, possibly by facilitating transmembrane pore closure. In this way, serum components or synthetic polymers can be used to increase intracellular delivery by nanoparticle-mediated photoporation while maintaining high cell viability.


Assuntos
Citoproteção , Sistemas de Liberação de Medicamentos , Espaço Intracelular/química , Luz , Nanopartículas/química , Soro/química , Carboximetilcelulose Sódica/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Citoproteção/efeitos da radiação , Fibroblastos/efeitos da radiação , Humanos , Lasers , Peso Molecular , Poloxâmero/química , Polietilenoglicóis/química , Fuligem/química , Viscosidade
19.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065474

RESUMO

Obesity-induced adipose tissue dysfunction and disorders of glycolipid metabolism have become a worldwide research priority. Zfp217 plays a crucial role in adipogenesis of 3T3-L1 preadipocytes, but about its functions in animal models are not yet clear. To explore the role of Zfp217 in high-fat diet (HFD)-induced obese mice, global Zfp217 heterozygous knockout (Zfp217+/-) mice were constructed. Zfp217+/- mice and Zfp217+/+ mice fed a normal chow diet (NC) did not differ significantly in weight gain, percent body fat mass, glucose tolerance, or insulin sensitivity. When challenged with HFD, Zfp217+/- mice had less weight gain than Zfp217+/+ mice. Histological observations revealed that Zfp217+/- mice fed a high-fat diet had much smaller white adipocytes in inguinal white adipose tissue (iWAT). Zfp217+/- mice had improved metabolic profiles, including improved glucose tolerance, enhanced insulin sensitivity, and increased energy expenditure compared to the Zfp217+/+ mice under HFD. We found that adipogenesis-related genes were increased and metabolic thermogenesis-related genes were decreased in the iWAT of HFD-fed Zfp217+/+ mice compared to Zfp217+/- mice. In addition, adipogenesis was markedly reduced in mouse embryonic fibroblasts (MEFs) from Zfp217-deleted mice. Together, these data indicate that Zfp217 is a regulator of energy metabolism and it is likely to provide novel insight into treatment for obesity.


Assuntos
Metabolismo Energético/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Transativadores/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos Brancos/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/fisiopatologia , Animais , Dieta Hiperlipídica , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Termogênese/fisiologia , Ganho de Peso/fisiologia
20.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070360

RESUMO

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A2A and A2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A2A, A2B and PKA. As a result of Wnt/ß-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFß) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/ß-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.


Assuntos
Adenosina/farmacologia , Desoxiadenosinas/farmacologia , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular , Fibroblastos/patologia , Humanos , Pele/lesões , Pele/metabolismo , Pele/patologia , Cicatrização/efeitos dos fármacos , beta Catenina/metabolismo
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