Modelos avançados de pele humana 3D in vitro: epiderme bioimpressa e pele tricamada contendo hipoderme para avaliação de segurança e mimético de dermatite atópica nocaute de IL-13Rα2 para estudo da função barreira / Advanced in vitro 3D human skin models: bioprinted epidermis and trilayer skin containing hypodermis for safety assessment and atopic dermatitis mimetic with IL-13Rα2 knockout for barrier function study

A reconstrução de modelos avançados de pele tridimensional in vitro, que corresponda de forma mais fidedigna ao complexo microambiente da pele humana, depende da utilização de inovações tecnológicas e da adição de novos tipos celulares representativos da pele humana. Desta maneira, estes miméticos fornecem uma plataforma de alta relevância para estudos de fisiopatologia da pele, além de propiciar um sistema para a avaliação da segurança e eficácia de cosméticos e medicamentos alternativo ao uso de animais. Dessa maneira, o Capítulo I compara a performance de uma epiderme reconstruída humana (RHE) bioimpressa com a manual utilizando o teste in vitro de irritação cutânea descrito no guia OCDE número 439. Nossos resultados demonstram que ambos os modelos de pele exibiram morfologia estratificada e a função barreira epidérmica equivalente aos modelos validados. Nos testes de irritação in vitro, ambos modelos distinguiram corretamente as substâncias de referência, classificadas entre irritantes ou não-irritantes de acordo com o limiar de viabilidade de 50%. Esse resultado indica que a bioimpressora poderia ser de grande utilidade para a automação da reconstrução de modelos epidérmicos. O tecido hipodérmico possui importante papel na homeostase da pele humana. O Capítulo II aborda a reconstrução de uma pele tricamada, contendo a camada hipodérmica, além da epiderme e derme. Usando esferoides de adipócitos diferenciados in vitro, um modelo de pele tricamada em matriz de colágeno foi construído. Ao comparar este com a pele bicamada obtivemos maior expressão de loricrina e involucrina no modelo tricamada, indicando um potencial para maior função barreira, além de maior expressão de PPAR-γ. Testes de função barreira através da resistividade elétrica não demonstraram diferenças entre os modelos, mas a aplicação de SDS a 5 mg/ml por 18 horas induziu o aumento da viabilidade na pele tricamada. Além disso, após a aplicação de SDS a 2,5% para induzir uma irritação aguda, seguida de recuperação por 42h, obtivemos maior viabilidade na pele tricamada, indicando melhor recuperação pós-lesão irritativa induzida. A pele tricamada é promissora para estudos do metabolismo da pele humana e recuperação de lesões. A dermatite atópica (DA) é uma doença eczematosa de pele caracterizada por inflamação do tipo Th2 e alteração da barreira epidérmica. IL-13 e IL-4 são centrais no comprometimento da barreira epidérmica na DA. Entre os receptores de IL-13 em queratinócitos, o receptor IL-13Rα2, tem um papel controverso na alteração da barreira cutânea. O objetivo do Capítulo III foi estudar a deleção da expressão de IL-13Rα2 em RHE, que foram expostas a IL-4 e IL-13, e avaliadas conforme a expressão dos receptores e de proteínas alteradas na DA. As epidermes com knockout em IL-13Rα2 apresentaram redução da expressão de NELL2 (p<0,0021), tipicamente aumentadas na DA. Além disso, houve redução da expressão do receptor do IL-2Rγ. Assim, um possível papel de exacerbação da DA do receptor IL-13Rα2 deve ser estudado mais extensamente para ser caracterizado

The reconstruction of advanced three-dimensional in vitro skin models, which more reliably correspond to the complex microenvironment of human skin, depends on the use of technological innovations and the addition of new cell types representative of human skin.In this way, these mimetics provide a highly relevant platform for studies of skin pathophysiology, in addition to providing a system for evaluating the safety and efficacy of cosmetics and medicines alternative to animal use. In this way, Chapter I compares the performance of a bioprinted human reconstructed epidermis (RHE) with a manual one using the in vitro skin irritation test described in OECD guide number 439. Our results demonstrate that both skin models exhibited stratified morphology and the epidermal barrier function equivalent to validated models. In in vitro irritation tests, both models correctly distinguished the reference substances, classified as irritating or non-irritating according to the viability threshold of 50%. This result indicates that the bioprinter could be of great use for automating the reconstruction of epidermal models Hypodermic tissue plays an important role in the homeostasis of human skin. Chapter II addresses the reconstruction of a three-layer skin, containing the hypodermic layer, in addition to the epidermis and dermis. Using in vitro differentiated adipocyte spheroids, a trilayer skin model in collagen matrix was constructed. When comparing this with bilayer skin, we obtained greater expression of loricrin and involucrin in the trilayer model, indicating a potential for greater barrier function, in addition to greater expression of PPAR-γ . Barrier function tests using electrical resistivity did not demonstrate differences between the models, but the application of SDS at 5 mg/ml for 18 hours induced an increase in viability in the three-layer skin. Furthermore, after applying 2.5% SDS to induce acute irritation, followed by recovery for 42 hours, we obtained greater viability in the three-layer skin, indicating better recovery after induced irritant injury. Trilayer skin holds promise for studies of human skin metabolism and injury recovery. Atopic dermatitis (AD) is an eczematous skin disease characterized by Th2-type inflammation and alteration of the epidermal barrier. IL-13 and IL-4 are central to the impairment of the epidermal barrier in AD. Among the IL-13 receptors on keratinocytes, the IL-13Rα2 receptor has a controversial role in altering the skin barrier. The objective of Chapter III was to study the deletion of IL-13Rα2 expression in RHE, which were exposed to IL-4 and IL-13, and evaluated according to the expression of receptors and proteins altered in AD. Epidermis with IL-13Rα2 knockout showed reduced NELL2 expression (p<0.0021), typically increased in AD. Furthermore, there was a reduction in the expression of the IL-2Rγ receptor. Therefore, a possible AD exacerbation role of the IL-13Rα2 receptor should be studied more extensively to be characterized

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

Protective effects of liquiritin on UVB-induced skin damage in SD rats.

Overexposure to ultraviolet B (UVB) rays can cause damage to the skin. Liquiritin has a variety of pharmacological effects, such as anti-inflammatory and antioxidant. In the present study, the effect of liquiritin on UVB irradiated rat skin was investigated. Results showed that UVB irradiation caused erythema and wrinkles on the skin surface, as well as thickening and loss of elasticity of the epidermis and a significant increase in the level of ROS in the skin tissue. At the same time, western blot detected an increase in nuclear factor kappa-B (NF-κB) and matrix metalloproteinases (MMPs) and Elisa also detected an increase in pro-inflammatory factors. Therefore, we hypothesized that UVB irradiation-induced damage is associated with inflammation. Interestingly, application of liquiritin to exposed skin of rats reduced the increase in ROS, pro-inflammatory factors, and MMPs caused by UVB irradiation and increased the levels of Sirtuin3 (SIRT3) and Collagen α1. In addition, after intraperitoneal injection of the SIRT3 inhibitor 3-TYP in rats, the protective effect of liquiritin against UVB damage was found to be diminished. These results suggested that promotion of SIRT3 with liquiritin inhibits UVB-induced production of pro-inflammatory mediators, possibly acting through the SIRT3/ROS/NF-κB pathway. In conclusion, this study suggests that liquiritin is an effective drug candidate for the prevention of UVB damage.

Concentration of collagen fibers in the musculature of broiler chickens fed with cupuacu seed by-product / Concentração de fibras colágenas na musculatura de frangos alimentados com farinha de cupuaçu

In this essay, we quantify the concentration of collagen fibers in broiler chickens exposed to increasing concentrations of cupuacu seed by-product. Collection of material was carried out in five chickens per treatment at 70 days old in the groups: control, 5% and 10% inclusion of cupuacu seed by-product. Fragments of Thoracic Pectoralis (PT) and Iliotibial lateralis (ITL) muscles were prepared for light and electronic microscopy. The amount of collagen fibers in the muscle groups was 1.08±0.61% in the PTC group; 6.24±2.58% in PT5% and 7.30±2.75% in PT10%. In the Iliotibial Lateralis groups, the results were 6.96±3.14% in the ITLC; 7.43±4.22% in the ITL5% and 8.66±2.35% in ITL10%. The amount of collagen fibers in the ITL5% and ITL10% groups showed no significant statistical difference. However, when compared to the ILTC group, there was a significant statistical difference. The PT muscle responds to standard nutritional changes, unlike the ILT muscle, which requires a high-nutrient formulation. The use of 5% cupuacu seed by-product has proven to be a viable alternative source of animal feed, as it promotes an increase in the concentrations of collagen fibers in the musculature of broiler chickens and is possibly the determining factor in meat texture.(AU)

Neste estudo, foram quantificadas as concentrações de fibras colágenas de frangos expostos a crescentes concentrações de farinha de cupuaçu. A coleta de material foi realizada em cinco animais por tratamento, aos 70 dias de idade, nos grupos: controle, inclusão de 5% e de 10% de farinha de cupuaçu. Fragmentos dos músculos peitoral torácico (PT) e iliotibial lateral (ITL) foram preparados para microscopia de luz e eletrônica. A quantidade de fibras colágenas nos grupos foi: 1,08±0,61% no grupo PTC; 6,24±2,58% em PT5% e 7,30±2,75% em PT10%. Nos grupos iliotibial lateral, os resultados foram: 6,96±3,14% no ITLC; 7,43±4,22% no ITL5% e 8,66±2,35% em ITL10%. A quantidade de fibras colágenas nos grupos ITL5% e ITL10% não apresentou diferença estatística significativa. No entanto, quando comparados ao grupo ILTC, houve diferença estatística significativa. O músculo PT responde a mudanças nutricionais padrão, ao contrário do músculo ILT, que requer alta formulação nutricional. O uso de 5% de farinha de cupuaçu provou ser uma fonte alternativa viável de alimentação animal, pois promove um aumento nas concentrações de fibras de colágeno na musculatura de frangos de corte e é possivelmente um fator determinante na textura da carne.(AU)

Wound healing effects of nanoemulsion containing clove essential oil.

The aim of this study was to investigate the wound healing effects of clove oil (CO) via its encapsulation into nanoemulsion. Optimized nanoemulsion (droplet size of 29.10 nm) was selected for wound healing investigation, collagen determination, and histopathological examination in rats. Optimized nanoemulsion presented significant would healing effects in rats as compared to pure CO. Nanoemulsion also presented significant enhancement in leucine content (0.61 mg/g) as compared to pure CO (0.50 mg/g) and negative control (0.31 mg/g). Histopathology of nanoemulsion treated rats showed no signs of inflammatory cells. These results suggested that nanoemulsion of CO was safe and nontoxic.

Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats.

Tributyltin chloride (TBT) is an organometallic pollutant that is used as a biocide in antifouling paints. TBT induces several toxic and endocrine-disrupting effects. However, studies evaluating the effects of TBT on renal function are rare. This study demonstrates that TBT exposure is responsible for improper renal function as well as the development of abnormal morphophysiology in mammalian kidneys. Female rats were treated with TBT, and their renal morphophysiology was assessed. Morphophysiological abnormalities such as decreased glomerular filtration rate and increased proteinuria levels were observed in TBT rats. In addition, increases in inflammation, collagen deposition and α-smooth muscle actin (α-SMA) protein expression were observed in TBT kidneys. A disrupted cellular redox balance and apoptosis in kidney tissue were also observed in TBT rats. TBT rats demonstrated reduced serum estrogen levels and estrogen receptor-α (ERα) protein expression in renal cortex. Together, these data provide in vivo evidence that TBT is toxic to normal renal function and that these effects may be associated with renal histopathology complications, such as inflammation and fibrosis.

MicroRNA-9 regulates cardiac fibrosis by targeting PDGFR-ß in rats.

The proliferation of cardiac fibroblasts (CFs) and excessive deposition of extracellular matrix (ECM) are the main pathological characteristics of cardiac fibrosis. In recent years, microRNAs (miRNAs) have been found to be a new kind of regulator in cardiac fibrosis. The purpose of this study was to investigate the role of microRNA-9 (miR-9) in the process of cardiac fibrosis and its mechanism. Treatment of cultured neonatal rat CFs with PDGF-BB or serum suppressed the expression of miR-9. Overexpression of miR-9 obviously inhibited neonatal rat CFs proliferation and collagen production as detected by MTT assays, qRT-PCR, and western blotting. The effects of miR-9 in CFs were abrogated by co-transfection with miR-9 inhibitors. Overexpression of miR-9 reduced the mRNA and protein levels of PDGFR-ßand its downstream protein, extracellular signal-regulated kinase (ERK) 1/2. Silencing PDGFR-ßby small interfering RNA mimicked the anti-fibrotic action of miR-9, whereas overexpression of PGDFR-ß canceled the effect of miR-9 in cultured CFs. Dual-luciferase reporter assays showed that PDGFR-ßwas a direct target of miR-9. Overexpression of miR-9 inhibited cardiac fibrosis by targeting PDGFR-ß, indicating that miR-9 might play a role in the treatment of cardiac fibrosis.

Self-Assembly and Collagen-Stimulating Activity of a Peptide Amphiphile Incorporating a Peptide Sequence from Lumican.

The self-assembly and bioactivity of a peptide amphiphile (PA) incorporating a 13-residue sequence derived from the last 13 amino acids of the C-terminus of lumican, C16-YEALRVANEVTLN, attached to a hexadecyl (C16) lipid chain have been examined. Lumican is a proteoglycan found in many types of tissue and is involved in collagen fibril organization. A critical aggregation concentration (cac) for the PA was determined through pyrene fluorescence measurements. The structure of the aggregates was imaged using electron microscopy, and twisted and curved nanotapes were observed. In situ small-angle X-ray scattering and fiber X-ray diffraction reveal that these tapes contain interdigitated bilayers of the PA molecules. FTIR and circular dichroism spectroscopy and fiber X-ray diffraction indicate that the lumican sequence in the PA adopts a ß-sheet secondary structure. Cell assays using human dermal fibroblasts show that below the cac the PA displays good biocompatibility and also stimulates collagen production over a period of 3 weeks, exceeding a 2-fold enhancement for several concentrations. Thus, this PA has promise in future biological applications, in particular, in tissue engineering.

Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis.

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukaemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to the Fli1 silencing-dependent collagen induction, the simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B-cell activation and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.

[Correction of a connective tissue dysplasia in the treatment of postoperative abdominal hernias].

There were analyzed the results of treatment of 112 patients, suffering postoperative abdominal hernia, in whom the anterior abdominal wall alloplasty was performed as well as postoperative pathogenetically substantiated complex therapy, taking into account the presence of a connective tissue dysplasia syndrome (CTDS) and the early and late postoperative complications prophylaxis. The peculiarities of postoperative period course and late follow-up results were studied up. Phenotypic features of CTDS were revealed in 53 (47.3%) patients, immunohistochemical features of a connective tissue dysplasia (a failed collagen type I and III ratio, manifested by increase of a collagen type III fibers quantity in 3 or more times) were revealed in 78 (69.6%) patients, in whom the processes of a collagen and its supermolecular formations synthesis were stimulated, using a magnesium orotate (Magnerot), which was prescribed in 1 g dose twice a day during 4 - 6 weeks. Application of composite nets, owing big pores, in a complex with a postoperative pathogenetically substantiated therapy conduction have positively influenced the disease course and the late follow-up results achieved.

The prevalence of the platelet glycoprotein IIIa Pl(A1/A2) polymorphism in three South African ethnic groups and its effect on platelet function.

INTRODUCTION: In South Africa coronary artery disease (CAD) is less common in African than Indian or white subjects. Although the association between CAD and metabolic factors have been well documented, the role of genetic factors is as yet poorly understood. Specific polymorphisms in the platelet membrane glycoprotein (GP) IIIa gene Pl(A1/A2), have been implicated in the development of CAD. METHODS: The prevalence of platelet GPIIIa (Pl(A1/A2)) polymorphisms and their effect on platelet function was determined in 313 Indian, 267 white and 227 African subjects with and without a history of CAD. RESULTS: In subjects without a history of CAD the frequency of the unfavourable Pl(A2) allele was 8.0%, 14.8% and 8.7% in the Indian, white and African populations respectively, with the frequency being significantly higher (p<0.05) in the white than both other groups. The frequency of the Pl(A2) allele was higher in subjects with (23.0%) than without (10.0%; p<0.0001) a history of CAD. Aggregation studies showed that platelets carrying the Pl(A2) allele were hypersensitive to the platelet aggregating agonists ADP and collagen and produced a higher amount of TXA(2) when stimulated with low concentrations of both these agonists. CONCLUSIONS: The positive association observed between the platelet GPIIIa Pl(A1/A2) polymorphism and platelet function suggests that the GPIIIa Pl(A2) allele may be a genetic factor that contributes to the risk of sudden death from myocardial infarction in the absence of known risk factors but it does not explain ethnic differences in the prevalence of CAD.

The reprolysin jararhagin, a snake venom metalloproteinase, functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling.

The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins.