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Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.
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Proteínas Filagrinas , Complexo de Endopeptidases do Proteassoma , Humanos , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
In the nanomedicine field, there is a need to widen the availability of nanovectors to compensate for the increasingly reported side effects of poly(ethene glycol). Nanovectors enabling cross-linking can further optimize drug delivery. Cross-linkable polyoxazolines are therefore relevant candidates to address these two points. Here we present the synthesis of coumarin-functionalized poly(2-alkyl-2-oxazoline) block copolymers, namely, poly(2-methyl-2-oxazoline)-block-poly(2-phenyl-2-oxazoline) and poly(2-methyl-2-oxazoline)-block-poly(2-butyl-2-oxazoline). The hydrophilic ratio and molecular weights were varied in order to obtain a range of possible behaviors. Their self-assembly after nanoprecipitation or film rehydration was examined. The resulting nano-objects were fully characterized by transmission electron microscopy (TEM), cryo-TEM, multiple-angle dynamic and static light scattering. In most cases, the formation of polymer micelles was observed, as well as, in some cases, aggregates, which made characterization more difficult. Cross-linking was performed under UV illumination in the presence of a coumarin-bearing cross-linker based on polymethacrylate derivatives. Addition of the photo-cross-linker and cross-linking resulted in better-defined objects with improved stability in most cases.
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Poliaminas , Polímeros , Sistemas de Liberação de Medicamentos , MicelasRESUMO
Congenital anomalies of the kidney and the urinary tract (CAKUT) are the first cause of chronic kidney disease in childhood. Several genetic and environmental origins are associated with CAKUT, but most pathogenic pathways remain elusive. Considering the amniotic fluid (AF) composition as a proxy for fetal kidney development, we analyzed the AF proteome from non-severe CAKUT (n = 19), severe CAKUT (n = 14), and healthy control (n = 22) fetuses using LC-MS/MS. We identified 471 significant proteins that discriminated the three AF groups with 81% precision. Among them, eight proteins independent of gestational age (CSPG4, LMAN2, ENDOD1, ANGPTL2, PRSS8, NGFR, ROBO4, PLS3) were associated with both the presence and the severity of CAKUT. Among those, five were part of a protein-protein interaction network involving proteins previously identified as being potentially associated with CAKUT. The actin-bundling protein PLS3 (plastin 3) was the only protein displaying a gradually increased AF abundance from control, via non-severe, to severe CAKUT. Immunohistochemistry experiments showed that PLS3 was expressed in the human fetal as well as in both the fetal and the postnatal mouse kidney. In zebrafish embryos, depletion of PLS3 led to a general disruption of embryonic growth including reduced pronephros development. In postnatal Pls3-knockout mice, kidneys were macroscopically normal, but the glomerular ultrastructure showed thickening of the basement membrane and fusion of podocyte foot processes. These structural changes were associated with albuminuria and decreased expression of podocyte markers including Wilms' tumor-1 protein, nephrin, and podocalyxin. In conclusion, we provide the first map of the CAKUT AF proteome that will serve as a reference for future studies. Among the proteins strongly associated with CAKUT, PLS3 did surprisingly not specifically affect nephrogenesis but was found as a new contributor in the maintenance of normal kidney function, at least in part through the control of glomerular integrity. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Líquido Amniótico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Anormalidades Urogenitais/metabolismo , Refluxo Vesicoureteral/metabolismo , Animais , Feminino , Feto , Humanos , Masculino , Camundongos , Proteoma , Proteômica , Peixe-ZebraRESUMO
Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.
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Dermatite Atópica , Infecções Estafilocócicas , Humanos , Dermatite Atópica/metabolismo , Staphylococcus aureus/metabolismo , Epiderme/metabolismo , Pele/metabolismo , Citocinas/metabolismo , Infecções Estafilocócicas/metabolismo , Higiene da PeleRESUMO
BACKGROUND: The pathogenesis of human atopic dermatitis (AD) is complex. Like humans, dogs develop spontaneous AD so this species could be a useful model of study. However, AD has been less characterised in dogs than in humans. OBJECTIVES: To compare the epidermis of normal and spontaneously atopic dogs at the functional and structural levels. ANIMALS: Six healthy and five atopic laboratory Beagle dogs. METHODS AND MATERIALS: Dogs were clinically characterised by general examination, Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) evaluation and trans-epidermal water loss (TWEL) measurement. Skin biopsies were taken from healthy skin from normal dogs and on nonlesional and lesional skin from atopic dogs. Samples were analysed using transmission electron microscopy (TEM). Cornified envelopes were extracted and examined for their visual aspects (smooth versus ruffled). RESULTS: CADESI-04 and TWEL were significantly higher in atopic dogs. Healthy and nonlesional skin could be distinguished from lesional skin by histopathological evaluation. TEM examination revealed abnormal morphology of the stratum corneum (SC) in atopic skin. The SC compactum corneocyte layer was larger. Thicker and wrinkled corneocytes were more prominent (P = 0.005) in the lesional skin. Similar changes were observed in the nonlesional skin, but less pronounced. The proportion of immature ruffled envelopes was increased in atopic samples (P < 0.05), both from lesional and nonlesional areas. CONCLUSIONS: The morphology of the SC was altered in the lesional and apparently nonlesional skin of spontaneously atopic dogs.
Assuntos
Dermatite Atópica , Doenças do Cão , Animais , Dermatite Atópica/veterinária , Cães , Células Epidérmicas , Epiderme , Microscopia Eletrônica de Transmissão/veterinária , PeleRESUMO
PURPOSE: The aim of the study was to evaluate organogel nanoparticles as a lipophilic vehicle to increase the oral bioavailability of poorly soluble compounds. Efavirenz (EFV), a Biopharmaceutical Classification System (BCS) Class II, was used as drug model. METHODS: Organogel nanoparticles loaded with EFV were formulated with sunflower oil, 12-hydroxystearic acid (HSA) and polyvinyl alcohol (PVA). Various parameters have been investigated in the current study such as (i) the release profile of organogel assessed by USP 4 cell flow dialysis, (ii) the impact of organogel on intestinal absorption, using Caco-2 cells as in vitro model and jejunum segments as ex vivo assay and (iii) the bioavailability of organogel following oral pharmacokinetic study. RESULTS: 250-300 nm spherical particles with a final concentration of 4.75 mg/mL drug loading were obtained, corresponding to a thousand fold increase in EFV solubility, combined to a very high encapsulation efficiency (>99.8%). Due to rapid diffusion, drug was immediately released from the nanoparticles. The biopharmaceutical evaluation on ex vivo jejunum segments demonstrated an increased absorption of EFV from organogel nanoparticles compare to a native EFV suspension. In vitro assays combining Caco-2 cell cultures with TEM and confocal microscopy demonstrated passive diffusion, while paracellular integrity and endocytosis activity remain expelled. Oral pharmacokinetics of EFV organogel nanoparticles improve oral bioavailability (Fr: 249%) and quick absorption compared to EFV suspension. CONCLUSION: Organogel nanoparticles increase the bioavailability of BCS Class II drugs. The main phenomena is simply oil transfer from the gelled particles through the cell membrane.
Assuntos
Benzoxazinas/química , Portadores de Fármacos/química , Géis/química , Nanocápsulas/química , Álcool de Polivinil/química , Ácidos Esteáricos/química , Óleo de Girassol/química , Alcinos , Animais , Benzoxazinas/administração & dosagem , Benzoxazinas/farmacocinética , Disponibilidade Biológica , Células CACO-2 , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclopropanos , Difusão , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Excipientes/química , Humanos , Absorção Intestinal , Masculino , Solubilidade , Suspensões/química , Distribuição TecidualRESUMO
Plastic pollution has become a worldwide concern. It was demonstrated that plastic breaks down to nanoscale particles in the environment, forming so-called nanoplastics. It is important to understand their ecological impact, but their structure is not elucidated. In this original work, we characterize the microstructure of oceanic polyethylene debris and compare it to the nonweathered objects. Cross sections are analyzed by several emergent mapping techniques. We highlight deep modifications of the debris within a layer a few hundred micrometers thick. The most intense modifications are macromolecule oxidation and a considerable decrease in the molecular weight. The adsorption of organic pollutants and trace metals is also confined to this outer layer. Fragmentation of the oxidized layer of the plastic debris is the most likely source of nanoplastics. Consequently the nanoplastic chemical nature differs greatly from plastics.
Assuntos
Polietileno , Poluentes Químicos da Água , Monitoramento Ambiental , Oceanos e Mares , Plásticos , ResíduosRESUMO
The global estimation of microplastic afloat in the ocean is only approximately 1% of annual global plastic inputs. This reflects fundamental knowledge gaps in the transformation, fragmentation, and fates of microplastics in the ocean. In order to better understand microplastic fragmentation we proceeded to a thorough physicochemical characterization of samples collected from the North Artlantic subtropical gyre during the sea campaign Expedition seventh Continent in May 2014. The results were confronted with a mathematical approach. The introduction of mass distribution in opposition to the size distribution commonly proposed in this area clarify the fragmentation pattern. The mathematical analysis of the mass distribution points out a lack of debris with mass lighter than 1 mg. Characterization by means of microscopy, microtomography, and infrared microscopy gives a better understanding of the behavior of microplastic at sea. Flat pieces of debris (2 to 5 mm in length) typically have one face that is more photodegraded (due to exposure to the sun) and the other with more biofilm, suggesting that they float in a preferred orientation. Smaller debris, with a cubic shape (below 2 mm), seems to roll at sea. All faces are evenly photodegraded and they are less colonized. The breakpoint in the mathematical model and the experimental observation around 2 mm leads to the conclusion that there is a discontinuity in the rate of fragmentation: we hypothesized that the smaller microplastics, the cubic ones mostly, are fragmented much faster than the parallelepipeds.
Assuntos
Monitoramento Ambiental , Plásticos , Utensílios Domésticos , Modelos Teóricos , ResíduosRESUMO
Polymersomes formed from amphiphilic block copolymers, such as poly(ethyleneoxide-b-ε-caprolactone) (PEO-b-PCL) or poly(ethyleneoxide-b-methylmethacrylate), were characterized by asymmetrical flow field-flow fractionation coupled with quasi-elastic light scattering (QELS), multi-angle light scattering (MALS), and refractive index detection, leading to the determination of their size, shape, and molecular weight. The method was cross-examined with more classical ones, like batch dynamic and static light scattering, electron microscopy, and atomic force microscopy. The results show good complementarities between all the techniques; asymmetrical flow field-flow fractionation being the most pertinent one when the sample exhibits several different types of population.
Assuntos
Fracionamento por Campo e Fluxo/instrumentação , Luz , Metilmetacrilato/química , Poliésteres/química , Espalhamento de Radiação , Tensoativos/química , Desenho de Equipamento , Tamanho da PartículaRESUMO
FLG is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it colocalized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis using benzyloxycarbonyl-pro-prolinal induced the accumulation of FLG monomers. Downregulation of PREP expression in reconstructed human epidermis using RNA interference confirmed the impact of PREP on FLG metabolism and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, western blotting, and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification such as loricrin. Consequently, at the functional level, the transepidermal electric resistance was drastically reduced.
RESUMO
Human platelet lysate (HPL), rich in growth factors, is increasingly recognized for its potential in tissue engineering and regenerative medicine. However, its use in liquid or gel form is constrained by limited stability and handling difficulties. This study aimed to develop dry and porous aerogels from HPL hydrogel using an environmentally friendly supercritical CO2-based shaping process, specifically tailored for tissue engineering applications. The aerogels produced retained their three-dimensional structure and demonstrated significant mechanical robustness and enhanced manageability. Impressively, they exhibited high water absorption capacity, absorbing 87% of their weight in water within 120 min. Furthermore, the growth factors released by these aerogels showed a sustained and favourable biological response in vitro. They maintained the cellular metabolic activity of fibroblasts (BALB-3T3) at levels akin to conventional culture conditions, even after prolonged storage, and facilitated the migration of human umbilical vein endothelial cells (HUVECs). Additionally, the aerogels themselves supported the adhesion and proliferation of murine fibroblasts (BALB-3T3). Beyond serving as excellent matrices for cell culture, these aerogels function as efficient systems for the delivery of growth factors. Their multifunctional capabilities position them as promising candidates for various tissue regeneration strategies. Importantly, the developed aerogels can be stored conveniently and are considered ready to use, enhancing their practicality and applicability in regenerative medicine.
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Because of the difficult challenges of nanopharmaceutics, the development of a variety of nanovectors is still highly desired. Photodynamic therapy, which uses a photosensitizer to locally produce reactive oxygen species to kill the undesired cells, is a typical example for which encapsulation has been shown to be beneficial. The present work describes the use of coumarin-functionalized polymeric nanovectors based on the self-assembly of amphiphilic poly(2-oxazoline)s. Encapsulation of pheophorbide a, a known PDT photosensitizer, is shown to lead to an increased efficiency compared to the un-encapsulated version. Interestingly, the presence of coumarin both enhances the desired photocytotoxicity and enables the crosslinking of the vectors. Various nanovectors are examined, differing by their size, shape and hydrophilicity. Their behaviour in PDT protocols on HCT-116 cells monolayers is described, the influence of their crosslinking commented. Furthermore, the formation of a protein corona is assessed.
Assuntos
Cumarínicos , Oxazóis , Fotoquimioterapia , Fármacos Fotossensibilizantes , Fotoquimioterapia/métodos , Humanos , Cumarínicos/química , Oxazóis/química , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Células HCT116 , Sobrevivência Celular/efeitos dos fármacos , Clorofila/análogos & derivados , Clorofila/química , Clorofila/farmacologia , Nanopartículas/química , Portadores de Fármacos/química , Polímeros/químicaRESUMO
Deimination is a post-translational modification catalyzed by a family of enzymes named peptidylarginine deiminases (PADs). PADs transform arginine residues of protein substrates into citrulline. Deimination has been associated with numerous physiological and pathological processes. In human skin, three PADs are expressed (PAD1-3). While PAD3 is important for hair shape formation, the role of PAD1 is less clear. To decipher the main role(s) of PAD1 in epidermal differentiation, its expression was down-regulated using lentivirus-mediated shRNA interference in primary keratinocytes and in three-dimensional reconstructed human epidermis (RHE). Compared to normal RHEs, down-regulation of PAD1 caused a drastic reduction in deiminated proteins. Whereas proliferation of keratinocytes was not affected, their differentiation was disturbed at molecular, cellular and functional levels. The number of corneocyte layers was significantly reduced, expression of filaggrin and cornified cell envelope components, such as loricrin and transglutaminases, was down-regulated, epidermal permeability increased and trans-epidermal-electric resistance diminished drastically. Keratohyalin granule density decreased and nucleophagy in the granular layer was disturbed. These results demonstrate that PAD1 is the main regulator of protein deimination in RHE. Its deficiency alters epidermal homeostasis, affecting the differentiation of keratinocytes, especially the cornification process, a special kind of programmed cell death.
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Previous work has shown increased expression of genes related to oxidative stress in nonlesional atopic dermatitis (ADNL) skin. Although mitochondria are key regulators of ROS production, their function in AD has never been investigated. Energy metabolism and the oxidative stress response were studied in keratinocytes (KCs) from patients with ADNL or healthy controls. Moreover, ADNL human epidermal equivalents were treated with tigecycline or MitoQ. We found that pyruvate and glucose were used as energy substrates by ADNL KCs. Increased mitochondrial oxidation of (very) long-chain fatty acids, associated with enhanced complexes I and II activities, was observed in ADNL KCs. Metabolomic analysis revealed increased tricarboxylic acid cycle turnover. Increased aerobic metabolism generated oxidative stress in ADNL KCs. ADNL human epidermal equivalents displayed increased mitochondrial function and an enhanced oxidative stress response compared with controls. Treatment of ADNL human epidermal equivalents with tigecycline or MitoQ largely corrected the AD profile, including high p-65 NF-κB, abnormal lamellar bodies, and cellular damage. Furthermore, we found that glycolysis supports but does not supersede mitochondrial metabolism in ADNL KCs. Thus, aerobic metabolism predominates in ADNL but leads to oxidative stress. Therefore, mitochondria could be a reservoir of potential therapeutic targets in atopic dermatitis.
Assuntos
Dermatite Atópica , Dermatite Atópica/genética , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Ácido Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tigeciclina/metabolismoRESUMO
The microsomal antiestrogen-binding site (AEBS) is a high-affinity membranous binding site for the antitumor drug tamoxifen that selectively binds diphenylmethane derivatives of tamoxifen such as PBPE and mediates their antiproliferative properties. The AEBS is a hetero-oligomeric complex consisting of 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase. High-affinity AEBS ligands inhibit these enzymes leading to the massive intracellular accumulation of zymostenol or 7-dehydrocholesterol (DHC), thus linking AEBS binding to the modulation of cholesterol metabolism and growth control. The aim of the present study was to gain more insight into the control of breast cancer cell growth by AEBS ligands. We report that PBPE and tamoxifen treatment induced differentiation in human breast adenocarcinoma cells MCF-7 as indicated by the arrest of cells in the G0-G1 phase of the cell cycle, the increase in the cell volume, the accumulation and secretion of lipids, and a milk fat globule protein found in milk. These effects were observed with other AEBS ligands and with zymostenol and DHC. Vitamin E abrogates the induction of differentiation and reverses the control of cell growth produced by AEBS ligands, zymostenol, and DHC, showing the importance of the oxidative processes in this effect. AEBS ligands induced differentiation in estrogen receptor-negative mammary tumor cell lines SKBr-3 and MDA-MB-468 but with a lower efficiency than observed with MCF-7. Together, these data show that AEBS ligands exert an antiproliferative effect on mammary cancer cells by inducing cell differentiation and growth arrest and highlight the importance of cholesterol metabolism in these effects.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Colesterol/metabolismo , Moduladores de Receptor Estrogênico/farmacologia , Microssomos/metabolismo , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Humanos , Ligantes , Lipídeos/química , Proteínas do Leite/química , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fatores de TempoRESUMO
Myosin Vb (Myo5b) is an unconventional myosin involved in the actin-dependent transport and tethering of intracellular organelles. In the epidermis, granular keratinocytes accumulate cytoplasmic lamellar bodies (LBs), secretory vesicles released at the junction with the stratum corneum that participate actively in the maintenance of the epidermal barrier. We have previously demonstrated that LB biogenesis is controlled by the Rab11a guanosine triphosphate hydrolase, known for its ability to recruit the Myo5b motor. In order to better characterize the molecular pathway that controls LB trafficking, we analyzed the role of F-actin and Myo5b in the epidermis. We demonstrated that LB distribution in granular keratinocytes was dependent on a dynamic F-actin cytoskeleton. Myo5b was shown to be highly expressed in granular keratinocytes and associated with corneodesmosin-loaded LB. In reconstructed human epidermis, Myo5b silencing led to epidermal barrier defects associated with structural alterations of the stratum corneum and a reduced pool of LB showing signs of disordered maturation. Myo5b depletion also disturbed the expression and distribution of both LB cargoes and junctional components, such as claudin-1, which demonstrates its action on both LB trafficking and junctional complex composition. Together, our data reveal the essential role of Myo5b in maintaining the epidermal barrier integrity.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Junções Íntimas/metabolismo , Células Cultivadas , Claudina-1/metabolismo , Epiderme/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/patologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
An amphiphilic polymer (CmPOX) based on poly(2-methyl-2-oxazoline) linked to a hydrophobic part composed of an aliphatic chain ending with a photo-active coumarin group has been synthesized. It exhibits the ability of forming small polymeric self-assemblies, typically of ca. 10 nm in size, which were characterized by TEM, cryo-TEM and DLS. The nanocarriers were further formulated to yield photo-crosslinked systems by dimerization of coumarin units of coumarin-functionalized poly(methyl methacrylate) (CmPMMA) and CmPOX. The formed vectors were used to encapsulate Pheophorbide a, a known photosensitizer for photodynamic therapy. Cytotoxicity as well as phototoxicity experiments performed in vitro on human tumor cells revealed the great potential of these nanovectors for photodynamic therapy.
Assuntos
Portadores de Fármacos/química , Interações Hidrofóbicas e Hidrofílicas , Oxazóis/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Polímeros/química , Clorofila/análogos & derivados , Clorofila/química , Clorofila/farmacologia , Células HCT116 , Humanos , Polimetil Metacrilato/químicaRESUMO
Septic shock is the most common cause of acute kidney injury (AKI), but the underlying mechanisms remain unclear and no targeted therapies exist. Lysophosphatidic acid (LPA) is a bioactive lipid which in vivo administration was reported to mitigate inflammation and injuries caused by bacterial endotoxemia in the liver and lung. The objective of the present study was to determine whether LPA can protect against sepsis-associated AKI. C57BL/6 mice were treated with LPA 18:1 (5 mg/kg, i.p.) 1 h before being injected with the endotoxin lipopolysaccharide (LPS), and AKI was evaluated after 24 h. LPA significantly decreased the elevation of plasma urea and creatinine caused by LPS. In the kidney, LPA pretreatment significantly reduced the upregulation of inflammatory cytokines (IL-6, TNFα, monocyte chemoattractant protein-1 (MCP-1)), and completely prevented downregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and upregulation of heme oxygenase-1 caused by LPS. LPA also prevented LPS-mediated alterations of the renal mitochondrial ultrastructure. In vitro pretreatment with LPA 18:1 significantly attenuated LPS-induced upregulation of the inflammatory cytokines (TNFα and MCP-1) in RAW264 macrophages. Moreover, in vivo LPS treatment lowered urinary LPA concentration and reduced LPA anabolic enzymes (autotaxin and acylglycerol kinase), and increased the LPA catalytic enzyme (lipid phosphate phosphatase 2) expression in the kidney cortex. In conclusion, exogenous LPA exerted a protective action against renal inflammation and injuries caused by bacterial endotoxemia. Moreover, LPS reduces the renal production of LPA suggesting that sepsis-associated AKI could be mediated, at least in part, by alleviation of the protective action of endogenous LPA.
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Injúria Renal Aguda/prevenção & controle , Lisofosfolipídeos/farmacologia , Injúria Renal Aguda/induzido quimicamente , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Endotoxinas , Inflamação/prevenção & controle , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Protetoras , Células RAW 264.7RESUMO
Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 micromol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3beta in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.
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Cálcio/metabolismo , Lipoproteínas VLDL/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Nucleares , Fosfatidilinositol 3-Quinases/metabolismo , Transcrição Gênica/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Calcineurina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Líquido Intracelular/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Fatores de Transcrição NFATC , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Most of the skin barrier function is attributable to the outermost layer of the epidermis, the stratum corneum, which is composed of flattened, anucleated cells called corneocytes surrounded by a lipid-enriched lamellar matrix. The composition of the stratum corneum is directly dependent on the underlying granular keratinocytes, which are the last living cells in the stratified epidermis. Many components present in the intercorneocyte matrix are delivered by the underlying granular keratinocytes through a secretion process dependent on lysosome-related organelles called lamellar bodies. Because of the importance of lamellar bodies in the maintenance of the epidermal barrier, the mechanisms regulating their biogenesis must be better understood. In this study, we show that the Rab11a GTPase is highly expressed in terminally differentiated keratinocytes, where it is partly associated with lamellar bodies. Rab11a silencing in three-dimensional in vitro reconstructed human epidermis induces a barrier defect, a decrease in the amount of lipid found in the stratum corneum, a reduction in lamellar body density and secretion areas in granular keratinocytes, and the mis-sorting of lamellar body cargoes being driven to the lysosomal degradation pathway. Our results highlight the importance of Rab11a-dependent regulation of lamellar body biogenesis in keratinocytes and consequently on epidermal barrier homeostasis.