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Adipose-derived mesenchymal stromal cells (AD-MSCs) are an essential issue in modern medicine. Extensive preclinical and clinical studies have shown that mesenchymal stromal/stem cells, including AD-MSCs, have specific properties (ability to differentiate into other cells, recruitment to the site of injury) of particular importance in the regenerative process. Ongoing research aims to elucidate factors supporting AD-MSC culture and differentiation in vitro. Angiopoietin-like proteins (ANGPTLs), known for their pleiotropic effects in lipid and glucose metabolism, may play a significant role in this context. Regeneration is a complex and dynamic process controlled by many factors. ANGPTL6 (Angiopoietin-related growth factor, AGF), among many activities modulated the biological activity of stem cells. This study examined the influence of synthesized AGF-derived peptides, designated as AGF9 and AGF27, on AD-MSCs. AGF9 and AGF27 enhanced the viability and migration of AD-MSCs and acted as a chemotactic factor for these cells. AGF9 stimulated chondrogenesis and lipid synthesis during AD-MSCs differentiation, influenced AD-MSCs cytokine secretion and modulated transcriptome for such basic cell activities as migration, transport of molecules, and apoptosis. The ability of AGF9 to modulate the biological activity of AD-MSCs warrants the consideration of this peptide a noteworthy therapeutic agent that deserves further investigation for applications in regenerative medicine.
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Tecido Adiposo , Proteínas Semelhantes a Angiopoietina , Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Humanos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas Semelhantes a Angiopoietina/metabolismo , Condrogênese/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Peptídeos/farmacologia , Movimento Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Citocinas/metabolismoRESUMO
Epidermal stem cells, located in the skin, together with keratinocytes are transplanted in regenerative therapies, e.g., for the treatment of burns or other wounds. Here, we describe the protocol of their enzymatic isolation from human skin. It includes separation of the epidermis form the dermis by incubation with dispase followed by cell isolation for epidermis by digestion with trypsin. Cell isolated with this method can be seeded on collagen IV-coated dishes. The methods of analysis of epidermal stem cells markers (e.g., CD71, CD29) with flow cytometry and RT-PCR are also included.
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Biomarcadores , Separação Celular , Colágeno Tipo IV , Células Epidérmicas , Citometria de Fluxo , Células-Tronco , Humanos , Citometria de Fluxo/métodos , Separação Celular/métodos , Células Epidérmicas/metabolismo , Células Epidérmicas/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Colágeno Tipo IV/metabolismo , Reação em Cadeia da Polimerase/métodos , Antígenos CD/metabolismo , Antígenos CD/genética , EndopeptidasesRESUMO
PURPOSE: The receptor-interacting protein kinase (RIPK4) has an oncogenic function in melanoma, regulates NF-κB and Wnt/ß-catenin pathways, and is sensitive to the BRAF inhibitors: vemurafenib and dabrafenib which lead to its decreased level. As its role in melanoma remains not fully understood, we examined the effects of its downregulation on the transcriptomic profile of melanoma. METHODS: Applying RNA-seq, we revealed global alterations in the transcriptome of WM266.4 cells with RIPK4 silencing. Functional partners of RIPK4 were evaluated using STRING and GeneMANIA databases. Cells with transient knockdown (via siRNA) and stable knockout (via CRISPR/Cas9) of RIPK4 were stimulated with TNF-α. The expression levels of selected proteins were assessed using Western blot, ELISA, and qPCR. RESULTS: Global analysis of gene expression changes indicates a complex role for RIPK4 in regulating adhesion, migration, proliferation, and inflammatory processes in melanoma cells. Our study highlights potential functional partners of RIPK4 such as BIRC3, TNF-α receptors, and MAP2K6. Data from RIPK4 knockout cells suggest a putative role for RIPK4 in modulating TNF-α-induced production of IL-8 and IL-6 through two distinct signaling pathways-BIRC3/NF-κB and p38/MAPK. Furthermore, increased serum TNF-α levels and the correlation of RIPK4 with NF-κB were revealed in melanoma patients. CONCLUSION: These data reveal a complex role for RIPK4 in regulating the immune signaling network in melanoma cells and suggest that this kinase may represent an alternative target for melanoma-targeted adjuvant therapy.
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Interleucina-6 , Interleucina-8 , Melanoma , Fator de Necrose Tumoral alfa , Humanos , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Melanoma/tratamento farmacológico , Interleucina-6/genética , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Linhagem Celular Tumoral , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
This study introduces a method for producing printable, thermosensitive bioink formulated from agarose (AG) and carbon dioxide-saturated chitosan (CS) hydrogels. The research identified medium molecular weight chitosan as optimal for bioink production, with a preferred chitosan hydrogel content of 40-60 %. Rheological analysis reveals the bioink's pseudoplastic behavior and a sol-gel phase transition between 27.0 and 31.5 °C. The MMW chitosan-based bioink showed also the most stable extrusion characteristic. The choice of chitosan for the production of bioink was also based on the assessment of the antimicrobial activity of the polymer as a function of its molecular weight and the degree of deacetylation, noting significant cell reduction rates for E. coli and S. aureus of 1.72 and 0.54 for optimal bioink composition, respectively. Cytotoxicity assessments via MTT and LDH tests confirm the bioink's safety for L929, HaCaT, and 46BR.1 N cell lines. Additionally, XTT proliferation assay proved the stimulating effect of the bioink on the proliferation of 46BR.1 N fibroblasts, comparable to that observed with Fetal Bovine Serum (FBS). FTIR spectroscopy confirms the bioink as a physical polymer blend. In conclusion, the CS/AG bioink demonstrates the promising potential for advanced spatial cell cultures in tissue engineering applications including skin regeneration.
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Dióxido de Carbono , Quitosana , Escherichia coli , Hidrogéis , Tinta , Sefarose , Quitosana/química , Quitosana/farmacologia , Dióxido de Carbono/química , Sefarose/química , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Animais , Escherichia coli/efeitos dos fármacos , Temperatura , Staphylococcus aureus/efeitos dos fármacos , Camundongos , Reologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Engenharia Tecidual/métodos , Antibacterianos/farmacologia , Antibacterianos/química , Fibroblastos/efeitos dos fármacosRESUMO
Non-healing wounds and skin losses constitute significant challenges for modern medicine and pharmacology. Conventional methods of wound treatment are effective in basic healthcare; however, they are insufficient in managing chronic wound and large skin defects, so novel, alternative methods of therapy are sought. Among the potentially innovative procedures, the use of skin substitutes may be a promising therapeutic method. Skin substitutes are a heterogeneous group of materials that are used to heal and close wounds and temporarily or permanently fulfill the functions of the skin. Classification can be based on the structure or type (biological and synthetic). Simple constructs (class I) have been widely researched over the years, and can be used in burns and ulcers. More complex substitutes (class II and III) are still studied, but these may be utilized in patients with deep skin defects. In addition, 3D bioprinting is a rapidly developing method used to create advanced skin constructs and their appendages. The aforementioned therapies represent an opportunity for treating patients with diabetic foot ulcers or deep skin burns. Despite these significant developments, further clinical trials are needed to allow the use skin substitutes in the personalized treatment of chronic wounds.
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Queimaduras , Pé Diabético , Pele Artificial , Humanos , Bioengenharia , Engenharia Biomédica , Queimaduras/terapiaRESUMO
BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.
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Biotecnologia , Celulose , Humanos , Proteínas Recombinantes de Fusão/metabolismo , Celulose/metabolismo , Proteínas Recombinantes/genética , HidróliseRESUMO
Hydrogels are three-dimensional polymer networks with hydrophilic properties. The modifiable properties of hydrogels and the structure resembling living tissue allow their versatile application. Therefore, increasing attention is focused on the use of hydrogels as bioinks for three-dimensional (3D) printing in tissue engineering. Bioprinting involves the fabrication of complex structures from several types of materials, cells, and bioactive compounds. Stem cells (SC), such as mesenchymal stromal cells (MSCs) are frequently employed in 3D constructs. SCs have desirable biological properties such as the ability to differentiate into various types of tissue and high proliferative capacity. Encapsulating SCs in 3D hydrogel constructs enhances their reparative abilities and improves the likelihood of reaching target tissues. In addition, created constructs can simulate the tissue environment and mimic biological signals. Importantly, the immunogenicity of scaffolds is minimized through the use of patient-specific cells and the biocompatibility and biodegradability of the employed biopolymers. Regenerative medicine is taking advantage of the aforementioned capabilities in regenerating various tissues- muscle, bones, nerves, heart, skin, and cartilage.
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Endothelial cells are a preferential target for SARS-CoV-2 infection. Previously, we have reported that vascular adenosine deaminase 1 (ADA1) may serve as a biomarker of endothelial activation and vascular inflammation, while ADA2 plays a critical role in monocyte and macrophage function. In this study, we investigated the activities of circulating ADA isoenzymes in patients 8 weeks after mild COVID-19 and related them to the parameters of inflammation and microvascular/endothelial function. Post-COVID patients revealed microvascular dysfunction associated with the changes in circulating parameters of endothelial dysfunction and inflammatory activation. Interestingly, serum total ADA and ADA2 activities were diminished in post-COVID patients, while ADA1 remained unchanged in comparison to healthy controls without a prior diagnosis of SARS-CoV-2 infection. While serum ADA1 activity tended to positively correspond with the parameters of endothelial activation and inflammation, sICAM-1 and TNFα, serum ADA2 activity correlated with IL-10. Simultaneously, post-COVID patients had lower circulating levels of ADA1-anchoring protein, CD26, that may serve as an alternative receptor for virus binding. This suggests that after the infection CD26 is rather maintained in cell-attached form, enabling ADA1 complexing. This study points to the possible role of ADA isoenzymes in cardiovascular complications after mild COVID-19.
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Adenosina Desaminase , COVID-19 , Doenças Vasculares , Humanos , COVID-19/metabolismo , Dipeptidil Peptidase 4 , Células Endoteliais , Inflamação , Isoenzimas , SARS-CoV-2RESUMO
Introduction: Chronic wounds are an increasing problem for health care all over the world. New treatment options for this illness are desired, especially antimicrobial agents. Silver nanoparticles (AgNPs) can be a potential substance that may be used in treatment of chronic wounds due to the growing antibiotic resistance. Aim: To synthetize silver nanoparticles that are stable, pure and effective against bacteria. Material and methods: The synthesis was conducted with chemical methods using different coating factors. The antistaphylococcal properties were analysed with the microdilution method to determine minimal inhibition concentrations (MIC) value. AgNPs were purified by dialysis. Moreover, keratinocyte cytotoxic properties of AgNPs were also assessed. Results: A method of synthesizing stable and efficient AgNPs has been developed. The type of the coating substance has a significant effect on AgNPs antimicrobial properties. Most of the silver nanoparticles, synthesized based on literature data, turned out to be durable during a few hours. This study has proven that depending on the coating factor, AgNPs stability ranges from 4 weeks to even 12 months. Unfortunately, the type of the stabilizer used also affects the cytotoxicity of AgNPs. It has been shown that dialysis is a substance purification method that is cheap, simple and easy to apply when dealing with high volume solutions. Conclusions: AgNPs could be an alternative to widely used antibiotics and disinfectants. Nevertheless, the introduction of those substances to health care requires detailed long-term research not only in the field of safe use, yet also durability and purity of AgNPs solutions used.
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Filaggrin (FLG) protein is indispensable for multiple aspects of the epidermal barrier function but its accumulation in a monomeric filaggrin form may initiate premature keratinocytes death; it is unclear how filaggrin levels are controlled before the formation of storing keratohyalin granules. Here we show that keratinocyte-secreted small extracellular vesicles (sEVs) may contain filaggrin-related cargo providing a route of eliminating excess filaggrin from keratinocytes; blocking of sEV release has cytotoxic effects on those cells. Filaggrin-containing sEVs are found in plasma in both healthy individuals and atopic dermatitis patients. Staphylococcus aureus (S. aureus) enhances packaging and secretion of filaggrin-relevant products within the sEVs for enhanced export via a TLR2-mediated mechanism which is also linked to the ubiquitination process. This filaggrin removal system, preventing premature keratinocyte death and epidermal barrier dysfunction, is exploited by S. aureus which promotes filaggrin elimination from the skin that could help safeguard bacterial growth.
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Vesículas Extracelulares , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Receptor 2 Toll-Like/metabolismo , Proteínas Filagrinas , Mortalidade Prematura , Vesículas Extracelulares/metabolismo , Queratinócitos/metabolismoRESUMO
Self-assembling peptides can be used for the regeneration of severely damaged skin. They can act as scaffolds for skin cells and as a reservoir of active compounds, to accelerate scarless wound healing. To overcome repeated administration of peptides which accelerate healing, we report development of three new peptide biomaterials based on the RADA16-I hydrogel functionalized with a sequence (AAPV) cleaved by human neutrophil elastase and short biologically active peptide motifs, namely GHK, KGHK and RDKVYR. The peptide hybrids were investigated for their structural aspects using circular dichroism, thioflavin T assay, transmission electron microscopy, and atomic force microscopy, as well as their rheological properties and stability in different fluids such as water or plasma, and their susceptibility to digestion by enzymes present in the wound environment. In addition, the morphology of the RADA-peptide hydrogels was examined with a unique technique called scanning electron cryomicroscopy. These experiments enabled us to verify if the designed peptides increased the bioactivity of the gel without disturbing its gelling processes. We demonstrate that the physicochemical properties of the designed hybrids were similar to those of the original RADA16-I. The materials behaved as expected, leaving the active motif free when treated with elastase. XTT and LDH tests on fibroblasts and keratinocytes were performed to assess the cytotoxicity of the RADA16-I hybrids, while the viability of cells treated with RADA16-I hybrids was evaluated in a model of human dermal fibroblasts. The hybrid peptides revealed no cytotoxicity; the cells grew and proliferated better than after treatment with RADA16-I alone. Improved wound healing following topical delivery of RADA-GHK and RADA-KGHK was demonstrated using a model of dorsal skin injury in mice and histological analyses. The presented results indicate further research is warranted into the engineered peptides as scaffolds for wound healing and tissue engineering.
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Hidrogéis , Sinais Direcionadores de Proteínas , Camundongos , Humanos , Animais , Hidrogéis/farmacologia , Hidrogéis/química , Peptídeos/farmacologia , Peptídeos/química , CicatrizaçãoRESUMO
Adipose-derived mesenchymal stromal cells (AD-MSCs) have been extensively studied in recent years. Their attractiveness is due to the ease of obtaining clinical material (fat tissue, lipoaspirate) and the relatively large number of AD-MSCs present in adipose tissue. In addition, AD-MSCs possess a high regenerative potential and immunomodulatory activities. Therefore, AD-MSCs have great potential in stem cell-based therapies in wound healing as well as in orthopedic, cardiovascular, or autoimmune diseases. There are many ongoing clinical trials on AD-MSC and in many cases their effectiveness has been proven. In this article, we present current knowledge about AD-MSCs based on our experience and other authors. We also demonstrate the application of AD-MSCs in selected pre-clinical models and clinical studies. Adipose-derived stromal cells can also be the pillar of the next generation of stem cells that will be chemically or genetically modified. Despite much research on these cells, there are still important and interesting areas to explore.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Tecido Adiposo , Diferenciação CelularRESUMO
There has been an increased interest in cell based therapies for a range of medical conditions in the last decade. This explosion in novel therapeutics research has led to the development of legislation specifically focused on cell and gene based therapies. In Europe, the European medicines agency (EMA) designates any medicines for human use which are based on genes, tissues, or cells as advanced therapy medicinal products or advanced therapy medicinal products (ATMPs). In this article we discuss the hurdles to widespread adoption of ATMPs in Europe, with a focus on regulatory T cells (Tregs). There are numerous barriers which must be overcome before mainstream adoption of Treg therapy becomes a reality. The source of the cells, whether to use autologous or allogenic cells, and the methods through which they are isolated and expanded, must all meet strict good manufacturing practice (GMP) standards to allow use of the products in humans. GMP compliance is costly, with the equipment and reagents providing a significant cost barrier and requiring specialized facilities and personnel. Conforming to the regulations set centrally by the EMA is difficult, and the different interpretations of the regulations across the various member states further complicates the regulatory approval process. The end products then require a complex and robust distribution network to ensure timely delivery of potentially life saving treatments to patients. In a European market whose logistics networks have been hammered by COVID and Brexit, ensuring rapid and reliable delivery systems is a more complex task than ever. In this article we will examine the impact of these barriers on the development and adoption of Tregs in Europe, and potential approaches which could facilitate more widespread use of Tregs, instead of its current concentration in a few very specialized centers.
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DNA methyltransferase inhibitor zebularine was proven to induce regeneration in the ear pinna in mice. We utilized a dorsal skin wound model to further evaluate this epigenetic inhibitor in wound healing. Full-thickness excisional wounds were made on the dorsum of 2 and 10-month-old healthy BALB/c and 3 and 8-month-old diabetic (db/db) mice, followed by topical or intraperitoneal zebularine delivery. Depending on the strain, age, dose, and delivery, the zebularine treatments either had no effect or accelerated or delayed wound closure. In principle, zebularine applied topically moderately promoted wound closure in the healthy but markedly delayed in the diabetic mice, which was in line with decreased viability of cultured keratinocytes from diabetic patients exposed to zebularine. The histological analysis revealed an improvement in the architecture of restored skin in zebularine-treated mice, manifested as a distinct layered pattern resembling panniculus carnosus. The finding corresponds with the zebularine-mediated activation of the Wnt5a gene, an essential regulator of Wnt signaling, the pathway involved in hair follicle development, the process which in turn is connected with regenerative skin healing. Although zebularine did not remarkably accelerate wound healing, zebularine and other epigenetic inhibitors deserve further testing as potential drugs to improve the quality of restored skin.
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Diabetes Mellitus Experimental , Camundongos , Animais , Diabetes Mellitus Experimental/patologia , Cicatrização , Pele/patologia , Epigênese GenéticaRESUMO
Candida albicans (C. albicans) infection is a potential complication in the individuals with atopic dermatitis (AD) and can affect clinical course of the disease. Here, using primary keratinocytes we determined that atopic milieu promotes changes in the interaction of small extracellular vesicles (sEVs) with dendritic cells and that this is further enhanced by the presence of C. albicans. sEV uptake is largely dependent on the expression of glycans on their surface; modelling of the protein interactions indicated that recognition of this pathogen through C. albicans-relevant pattern recognition receptors (PRRs) is linked to several glycosylation enzymes which may in turn affect the expression of sEV glycans. Here, significant changes in the surface glycosylation pattern, as determined by lectin array, could be observed in sEVs upon a combined exposure of keratinocytes to AD cytokines and C. albicans. This included enhanced expression of multiple types of glycans, for which several dendritic cell receptors could be proposed as binding partners. Blocking experiments showed predominant involvement of the inhibitory Siglec-7 and -9 receptors in the sEV-cell interaction and the engagement of sialic acid-containing carbohydrate moieties on the surface of sEVs. This pointed on ST6 ß-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 ß,3-Galactosyltransferase 1 (C1GALT1) as potential enzymes involved in the process of remodelling of the sEV surface glycans upon C. albicans exposure. Our results suggest that, in combination with atopic dermatitis milieu, C. albicans promotes alterations in the glycosylation pattern of keratinocyte-derived sEVs to interact with inhibitory Siglecs on antigen presenting cells. Hence, a strategy aiming at this pathway to enhance antifungal responses and restrict pathogen spread could offer novel therapeutic options for skin candidiasis in AD.
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Candidíase , Dermatite Atópica , Vesículas Extracelulares , Candida albicans , Glicosilação , Humanos , Queratinócitos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
The alarming raise of multi-drug resistance among human microbial pathogens makes the development of novel therapeutics a priority task. In contrast to conventional antibiotics, antimicrobial peptides (AMPs), besides evoking a broad spectrum of activity against microorganisms, could offer additional benefits, such as the ability to neutralize toxins, modulate inflammatory response, eradicate bacterial and fungal biofilms or prevent their development. The latter properties are of special interest, as most antibiotics available on the market have limited ability to diffuse through rigid structures of biofilms. Lipidation of AMPs is considered as an effective approach for enhancement of their antimicrobial potential and in vivo stability; however, it could also have undesired impact on selectivity, solubility or the aggregation state of the modified peptides. In the present work, we describe the results of structural modifications of compounds designed based on cationic antimicrobial peptides DK5 and CAR-PEG-DK5, derivatized at their N-terminal part with fatty acids with different lengths of carbon chain. The proposed modifications substantially improved antimicrobial properties of the final compounds and their effectiveness in inhibition of biofilm development as well as eradication of pre-formed 24 h old biofilms of Candida albicans and Staphylococcus aureus. The most active compounds (C5-DK5, C12-DK5 and C12-CAR-PEG-DK5) were also potent against multi-drug resistant Staphylococcus aureus USA300 strain and clinical isolates of Pseudomonas aeruginosa. Both experimental and in silico methods revealed strong correlation between the length of fatty acid attached to the peptides and their final membranolytic properties, tendency to self-assemble and cytotoxicity.
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Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Estabilidade de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Análise Espectral , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Technological developments in the field of biologically active peptide applications in medicine have increased the need for new methods for peptide delivery. The disadvantage of peptides as drugs is their low biological stability. Recently, great attention has been paid to self-assembling peptides that can form fibrils. Such a formulation makes bioactive peptides more resistant to enzymatic degradation and druggable. Peptide fibrils can be carriers for peptides with interesting biological activities. These features open up prospects for using the peptide fibrils as long-acting drugs and are a valid alternative to conventional peptidic therapies. In our study, we designed new peptide scaffolds that are a hybrid of three interconnected amino acid sequences and are: pro-regenerative, cleavable by neutrophilic elastase, and fibril-forming. We intended to obtain peptides that are stable in the wound environment and that, when applied, would release a biologically active sequence. Our studies showed that the designed hybrid peptides show a high tendency toward regular fibril formation and are able to release the pro-regenerative sequence. Cytotoxicity studies showed that all the designed peptides were safe, did not cause cytotoxic effects and revealed a pro-regenerative potential in human fibroblast and keratinocyte cell lines. In vivo experiments in a dorsal skin injury model in mice indicated that two tested peptides moderately promote tissue repair in their free form. Our research proves that peptide fibrils can be a druggable form and a scaffold for active peptides.
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Portadores de Fármacos/química , Peptídeos/química , Peptídeos/farmacologia , Alicerces Teciduais/química , Cicatrização/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Fibroblastos , Humanos , Queratinócitos , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Proteólise , Medicina Regenerativa , Análise EspectralRESUMO
Wound healing complications affect thousands of people each year, thus constituting a profound economic and medical burden. Chronic wounds are a highly complex problem that usually affects elderly patients as well as patients with comorbidities such as diabetes, cancer (surgery, radiotherapy/chemotherapy) or autoimmune diseases. Currently available methods of their treatment are not fully effective, so new solutions are constantly being sought. Cell-based therapies seem to have great potential for use in stimulating wound healing. In recent years, much effort has been focused on characterizing of adipose-derived mesenchymal stromal cells (AD-MSCs) and evaluating their clinical use in regenerative medicine and other medical fields. These cells are easily obtained in large amounts from adipose tissue and show a high proregenerative potential, mainly through paracrine activities. In this review, the process of healing acute and nonhealing (chronic) wounds is detailed, with a special attention paid to the wounds of patients with diabetes and cancer. In addition, the methods and technical aspects of AD-MSCs isolation, culture and transplantation in chronic wounds are described, and the characteristics, genetic stability and role of AD-MSCs in wound healing are also summarized. The biological properties of AD-MSCs isolated from subcutaneous and visceral adipose tissue are compared. Additionally, methods to increase their therapeutic potential as well as factors that may affect their biological functions are summarized. Finally, their therapeutic potential in the treatment of diabetic and oncological wounds is also discussed.
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Tecido Adiposo , Células-Tronco Mesenquimais , Idoso , Humanos , Medicina Regenerativa , Células Estromais , CicatrizaçãoRESUMO
BACKGROUND: Although Mycophenolic Acid (MPA) is applied as prodrugs in clinic as an immunosuppressant, it also possesses anticancer activity. MPA acts as Inosine-5'-Monophosphate Dehydrogenase (IMPDH) inhibitor, where the carboxylic group at the end of the side chain interacts with Ser 276 of the enzyme via hydrogen bonds. Therefore, MPA derivatives with other polar groups indicated high inhibition too. On the other hand, potent anticancer agents like dacarbazine and cisplatin give numerous side-effects. OBJECTIVE: Based on the literature data, MPA derivatives should be explored towards anticancer properties. Conversion of the carboxylic group of MPA to amide could maintain antiproliferative activity. Therefore, we decided to investigate several amino acid and peptide derivatives of MPA against chosen cancer cell lines in vitro. METHODS: Amides of MPA hold threonine and arginine amino acid unit. These amino acid derivatives were tested as L and D enantiomers and both in free acid and methyl esters forms. Additionally, MPA was modified with tuftsin or retro-tuftsin as biologically active peptides, which could act as a drug carrier. RESULTS: Amino acid and peptide derivatives of MPA were investigated in vitro as potential anticancer agents on cell lines: Ab melanoma, A375 melanoma and SHSY5Y neuroblastoma. The activity of the tested compounds was compared to parent MPA and known chemotherapeutics: dacarbazine and cisplatin. CONCLUSION: Amino acid moiety and the sequence of amino acids in the peptide part influenced observed activity. The most active amino acid MPA analogues occurred to be D and L-threonine derivatives as methyl esters, probably due to better cell membrane penetration.
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Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Ácido Micofenólico/farmacologia , Peptídeos/farmacologia , Aminoácidos/síntese química , Aminoácidos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Ácido Micofenólico/síntese química , Ácido Micofenólico/química , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Antimicrobial peptides (AMPs) constitute a promising tool in the development of novel therapeutic agents useful in a wide range of bacterial and fungal infections. Among the modifications improving pharmacokinetic and pharmacodynamic characteristics of natural AMPs, an important role is played by lipidation. This study focuses on the newly designed and synthesized lipopeptides containing multiple Lys residues or their shorter homologues with palmitic acid (C16) attached to the side chain of a residue located in the center of the peptide sequence. The approach resulted in the development of lipopeptides representing a model of surfactants with two polar headgroups. The aim of this study is to explain how variations in the length of the peptide chain or the hydrocarbon side chain of an amino acid residue modified with C16, affect biological functions of lipopeptides, their self-assembling propensity, and their mode of action.