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1.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673372

RESUMO

One of the most severe effects of coronavirus disease 2019 (COVID-19) is lung disorders such as acute respiratory distress syndrome. In the absence of effective treatments, it is necessary to search for new therapies and therapeutic targets. Platelets play a fundamental role in respiratory disorders resulting from viral infections, being the first line of defense against viruses and essential in maintaining lung function. The direct application of platelet lysate (PL) obtained from the platelet-rich plasma of healthy donors could help in the improvement of the patient due its anti-inflammatory, immunomodulatory, antifibrotic, and repairing effects. This work evaluates PL nebulization by analyzing its levels of growth factors and its biological activity on lung fibroblast cell cultures, besides describing a scientific basis for its use in this kind of pathology. The data of the work suggest that the molecular levels and biological activity of the PL are maintained after nebulization. Airway administration would allow acting directly on the lung tissue modulating inflammation and stimulating reparative processes on key structures such as the alveolocapillary barrier, improving the disease and sequels. The protocol developed in this work is a first step for the study of nebulized PL both in animal experimentation and in clinical trials.


Assuntos
Anti-Inflamatórios/farmacologia , Fatores Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Plasma Rico em Plaquetas , Adulto , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/imunologia , Plaquetas/imunologia , Linhagem Celular , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Masculino , Nebulizadores e Vaporizadores , Plasma Rico em Plaquetas/imunologia , Resultado do Tratamento
2.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669136

RESUMO

The transplantation of various immune cell types are promising approaches for the treatment of ischemic cardiovascular disease including myocardial infarction (MI) and peripheral arterial disease (PAD). Major limitation of these so-called Advanced Therapy Medicinal Products (ATMPs) is the ischemic microenvironment affecting cell homeostasis and limiting the demanded effect of the transplanted cell products. Accordingly, different clinical and experimental strategies have been evolved to overcome these obstacles. Here, we give a short review of the different experimental and clinical strategies to solve these issues due to ischemic cardiovascular disease.


Assuntos
Transplante de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Hematopoéticas/metabolismo , Isquemia/terapia , Infarto do Miocárdio/terapia , Neovascularização Fisiológica/efeitos dos fármacos , Doença Arterial Periférica/terapia , Animais , Doenças Cardiovasculares/terapia , Hipóxia Celular/fisiologia , Transplante de Células/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Microambiente Celular/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , MicroRNAs/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/imunologia , Neovascularização Fisiológica/imunologia , Doença Arterial Periférica/imunologia
3.
Nat Commun ; 12(1): 1055, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594058

RESUMO

mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Arginina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Cinética , Lisina/metabolismo , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação/efeitos dos fármacos
4.
Am J Physiol Heart Circ Physiol ; 320(4): H1646-H1656, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33635165

RESUMO

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and ß-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the ß-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.


Assuntos
Receptores de Apelina/agonistas , Apelina/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Apelina/análogos & derivados , Receptores de Apelina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Preparação de Coração Isolado , Isoproterenol , Ligantes , Masculino , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia
5.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540814

RESUMO

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21-32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33-46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states.


Assuntos
Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/metabolismo , RNA Longo não Codificante/análise , Aorta/citologia , Vasos Coronários/citologia , Citocinas/farmacologia , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Longo não Codificante/isolamento & purificação , RNA-Seq , Estresse Mecânico , Transcrição Genética/efeitos dos fármacos , Transcriptoma
6.
Am J Physiol Heart Circ Physiol ; 320(4): H1738-H1748, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33635166

RESUMO

Emerging evidence suggests the exercise pressor reflex is exaggerated in early stage type 1 diabetes mellitus (T1DM). Piezo channels may play a role in this exaggeration, as blocking these channels attenuates the exaggerated pressor response to tendon stretch in T1DM rats. However, tendon stretch constitutes a different mechanical and physiological stimuli than that occurring during muscle contraction. Therefore, the purpose of this study was to determine the contribution of Piezo channels in evoking the pressor reflex during an intermittent muscle contraction in T1DM. In unanesthetized decerebrate rats, we compared the pressor and cardioaccelerator responses to intermittent muscle contraction before and after locally injecting grammostola spatulata mechanotoxin 4 (GsMTx-4, 0.25 µM) into the hindlimb vasculature. Although GsMTx-4 has a high potency for Piezo channels, it has also been suggested to block transient receptor potential cation (TRPC) channels. We, therefore, performed additional experiments to control for this possibility by also injecting SKF 96365 (10 µM), a TRPC channel blocker. We found that local injection of GsMTx-4, but not SKF 96365, attenuated the exaggerated peak pressor (ΔMAP before: 33 ± 3 mmHg, after: 22 ± 3 mmHg, P = 0.007) and pressor index (ΔBPi before: 668 ± 91 mmHg·s, after: 418 ± 81 mmHg·s, P = 0.021) response in streptozotocin (STZ) rats (n = 8). GsMTx-4 attenuated the exaggerated early onset pressor and the pressor response over time, which eliminated peak differences as well as those over time between T1DM and healthy controls. These data suggest that Piezo channels are an effective target to normalize the exercise pressor reflex in T1DM.NEW & NOTEWORTHY This is the first study to demonstrate that blocking Piezo channels is effective in ameliorating the exaggerated exercise pressor reflex evoked by intermittent muscle contraction, commonly occurring during physical activity, in T1DM. Thus, these findings suggest Piezo channels may serve as an effective therapeutic target to reduce the acute and prolonged cardiovascular strain that may occur during dynamic exercise in T1DM.


Assuntos
Sistema Nervoso Autônomo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Sistema Cardiovascular/inervação , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Moduladores de Transporte de Membrana/farmacologia , Contração Muscular , Músculo Esquelético/inervação , Reflexo Anormal/efeitos dos fármacos , Venenos de Aranha/farmacologia , Animais , Sistema Nervoso Autônomo/metabolismo , Sistema Nervoso Autônomo/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Masculino , Condicionamento Físico Animal , Ratos Sprague-Dawley , Fatores de Tempo
7.
BMC Complement Med Ther ; 21(1): 66, 2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602182

RESUMO

BACKGROUND: High glucose (HG)-induced reactive oxygen species (ROS) overproduction impairs angiogenesis that is one pivotal factor of wound healing process. Angiogenesis impairment induces delayed wound healing, whereby it eventually leads to amputation in cases of poorly controlled diabetes with diabetic ulceration. Porcine placenta extract (PPE) is a natural waste product that comprises plenty of bioactive agents including growth factors and antioxidants. It was reported as an effective compound that prevents ROS generation. The goal of this study was to investigate the in vitro effect of PPE on HG-induced ROS-mediated angiogenesis impairment. METHODS: Primary endothelial cells (HUVECs) and endothelial cell line (EA.hy926) were treated with HG in the presence of PPE. The endothelial cells (ECs) viability, intracellular ROS generation, migration, and angiogenesis were determined by MTT assay, DCFDA reagent, wound healing assay, and tube formation assay, respectively. Additionally, the molecular mechanism of PPE on HG-induced angiogenesis impairment was investigated by Western blot. The angiogenic growth factor secretion was also investigated by the sandwich ELISA technique. RESULTS: HG in the presence of PPE significantly decreased intracellular ROS overproduction compared to HG alone. HG in the presence of PPE significantly increased ECs viability, migration, and angiogenesis compared to HG alone by showing recovery of PI3K/Akt/ERK1/2 activation. HG in the presence of PPE also decreased ECs apoptosis compared to HG alone by decreasing p53/Bax/cleaved caspase 9/cleaved caspase 3 levels and increasing Bcl 2 level. CONCLUSION: PPE attenuated HG-induced intracellular ROS overproduction that improved ECs viability, proliferation, migration, and angiogenesis by showing recovery of PI3K/Akt/ERK1/2 activation and inhibition of ECs apoptosis. This study suggests PPE ameliorated HG-induced ROS-mediated angiogenesis impairment, whereby it potentially provides an alternative treatment for diabetic wounds.


Assuntos
Produtos Biológicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Glucose/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Placenta/química , Suínos , Cicatrização/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Produtos Biológicos/química , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
8.
ACS Appl Mater Interfaces ; 13(2): 2165-2178, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33400482

RESUMO

Oxidative damage to cells from metabolites at a wound site is one of the trickiest factors inhibiting tissue regeneration, especially with bulk damage. In addition, an excessive inflammatory reaction by the body at the wound site can make it even worse. How to scavenge the reactive oxygen species (ROS) produced from metabolism and inflammatory reactions has become a critical issue in tissue engineering. Here, we utilize the natural bioactive small molecules l-arginine and l-phenylalanine and the growth factor inositol to synthesize a branched poly(ester amide) (BPEA) to fabricate BPEA nanocapsules for vitamin E delivery at wound sites. BPEA nanocapsules loaded with vitamin E (BPEA@VE NCs) could protect cells from both extracellular and intracellular damage by scavenging ROS. Simultaneously, the inflammatory reaction could also be downregulated, benefiting from the introduction of l-arginine. Furthermore, the biodegradation products of BPEA are natural metabolites of the body, such as amino acids and growth factors, guaranteeing the biocompatibility of the BPEA@VE NCs. The protective ability of the BPEA@VE NCs was also investigated in vivo for accelerated wound healing. All the results indicate that the BPEA@VE NCs have promising potential for the modulation of the local microenvironment in tissue engineering for excellent antioxidative and anti-inflammatory properties.


Assuntos
Aminoácidos/administração & dosagem , Antioxidantes/administração & dosagem , Inositol/administração & dosagem , Nanocápsulas/química , Vitamina E/administração & dosagem , Cicatrização/efeitos dos fármacos , Aminoácidos/farmacologia , Aminoácidos/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Arginina/administração & dosagem , Arginina/farmacologia , Arginina/uso terapêutico , Células Endoteliais da Veia Umbilical Humana , Humanos , Inositol/farmacologia , Inositol/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Masculino , Camundongos , Células NIH 3T3 , Fenilalanina/administração & dosagem , Fenilalanina/farmacologia , Fenilalanina/uso terapêutico , Poliésteres/química , Células RAW 264.7 , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Engenharia Tecidual , Vitamina E/farmacologia , Vitamina E/uso terapêutico
9.
Life Sci ; 268: 118932, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33400933

RESUMO

The sophisticated chain of cellular and molecular episodes during wound healing includes cell migration, cell proliferation, deposition of extracellular matrix, and remodelling and are onerous to replicate. Encapsulation of growth factors (GFs) and Stem cell-based (SCs) has been proclaimed to accelerate healing by transforming every phase associated with wound healing to enhance skin regeneration. Therapeutic application of mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (PSCs) provides aid in wound fixing, tissue integrity restoration and function of impaired tissue. Several scientific studies have established the essential role GFs in wound healing and their reduced degree in the chronic wound. The overall limitation includes half-life, unfriendly microhabitat abundant with protease, and inadequate delivery approaches results in decreased delivery of effective amounts in a suitable time-based fashion. Advancements in the area of reformative medicine as well as tissue engineering have offered techniques competent of dispensing SCs and GFs in site-oriented manner. The progress in nanotechnology-based approaches attracts researcher to study and evaluate the potential of this SCs and GFs based therapy in chronic wounds. These techniques embrace the polymeric regime viz., nano-formulations, hydrogels, liposomes, scaffolds, nanofibers, metallic nanoparticles, lipid-based nanoparticles and dendrimers that have established better retort through targeting tissues when GFs and SCs are transported via these humans made devices. Assumed the current problems, improvements in delivery approaches and difficulties offered by chronic wounds, we hope to show that encapsulation of SCs and GFs loaded nanoformulations therapies is the rational next step in improving wound care.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Células-Tronco , Engenharia Tecidual/métodos , Cicatrização/fisiologia , Animais , Diferenciação Celular , Colágeno/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Matriz Extracelular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Neovascularização Fisiológica , Cicatrização/efeitos dos fármacos
10.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430095

RESUMO

An essential requirement for the success of in vitro maturation (IVM) of the oocyte is to provide an optimal microenvironment similar to in vivo conditions. Recently, somatic cell-based coculture or supplementation of a conditioned medium during IVM has been performed to obtain better quality of oocytes, because they mimic the in vivo reproductive tract by secreting paracrine factors. In this study, human adipose-derived stem cells (ASC) and their conditioned medium (ASC-CM) were applied to IVM of porcine oocytes to evaluate the effectiveness of ASC on oocyte development and subsequent embryo development. In results, both ASC and ASC-CM positively influence on oocyte maturation and embryo development by regulating growth factor receptors (VEGF, FGFR, and IGFR), apoptosis (BCL2), cumulus expansion (PTGS2, HAS2, and TNFAIP6), and oocyte maturation-related genes (GDF9 and BMP15). In particular, the fluorescence intensity of GDF9 and BMP15 was markedly upregulated in the oocytes from the ASC-CM group. Furthermore, significantly high levels of growth factors/cytokine including VEGF, bFGF, IGF-1, IL-10, and EGF were observed in ASC-CM. Additionally, the ASC-CM showed active scavenging activity by reducing the ROS production in a culture medium. Consequently, for the first time, this study demonstrated the effect of human ASC-CM on porcine oocyte development and the alteration of mRNA transcript levels in cumulus-oocyte complexes.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/química , Animais , Blastocisto/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Células do Cúmulo/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oogênese/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Suínos
11.
Eur J Med Chem ; 211: 113091, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33338869

RESUMO

Cyclin-dependent kinase 9 (CDK9) is an increasingly important potential cancer treatment target. Nowadays, developing selective CDK9 inhibitors has been extremely challenging as its ATP-binding sites are similar with other CDKs. Here, we report that the CDK9 inhibitor BAY-1143572 is converted into a series of proteolysis targeting chimeras (PROTACs) which leads to several compounds inducing the degradation of CDK9 in acute myeloid leukemia cells at a low nanomolar concentration. In addition, the most potent PROTAC molecule B03 could inhibit cell growth more effectively than warhead alone, with little inhibition of other kinases. This enhanced antiproliferative activity is mediated by a slight increase in kinase inhibitory activity and an increase in the level of apoptosis induction. Moreover, B03 could induce the degradation of CDK9 in vivo. Our work provides evidence that B03 represents a lead for further development and that CDK9 degradation is a potential valuable therapeutic strategy in acute myeloid leukemia.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proliferação de Células , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Inibidores de Proteínas Quinases/farmacologia
12.
Methods Mol Biol ; 2174: 19-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813242

RESUMO

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.


Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Antígeno AC133/análise , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Citometria de Fluxo , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Antígenos CD15/análise , Células-Tronco Neoplásicas/fisiologia , Células-Tronco Neurais/citologia
13.
J Surg Res ; 257: 468-476, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32896815

RESUMO

BACKGROUND: Donation after circulatory death donors (DCD) can expand the donor pool for heart transplantation, which primarily depends on brain death donors. Ischemia and reperfusion injury are inherent to the DCD process. We hypothesize that pharmacologic inhibition of interleukin-1 (IL-1) and/or IL-18 is protective to DCD hearts. MATERIALS AND METHODS: Following clinical protocol, in-situ ischemia time in control beating-heart donor (CBD) and DCD groups was less than 5 and 40 min, respectively. Wild type (WT) C57Bl6/j, IL-1 receptor type I knockout (IL-1RI-KO), and IL-18 KO mice were used. Hearts were reanimated for 90 min on a Langendorff system with Krebs-Henseleit buffer at 37°C, to assess physiologic parameters. Recombinant IL-1 receptor antagonist (IL-1Ra) and/or IL-18 binding protein (IL-18BP) were added to the Krebs-Henseleit buffer to inhibit IL-1 and/or the IL-18 signaling, respectively. RESULTS: Developed pressure and ± dP/dt were significantly impaired in the DCD-WT group compared to CBD-WT (P ≤ 0.05). Troponin release was higher in DCD-WT groups. Functional parameters were preserved, and troponin release was significantly less in the DCD knockout groups. Heart function was improved in DCD groups treated with IL-1Ra or IL-18BP compared to the DCD-WT group. CONCLUSIONS: Heart function was significantly impaired in the DCD-WT group compared to CBD-WT. Genetic deletion or pharmacologic blockade of IL-1 or IL-18 was protective to DCD hearts.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Obtenção de Tecidos e Órgãos , Animais , Morte , Avaliação Pré-Clínica de Medicamentos , Coração/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-18/antagonistas & inibidores , Interleucina-18/genética , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Masculino , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Distribuição Aleatória
14.
Methods Mol Biol ; 2225: 93-105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108659

RESUMO

In vivo wound healing models are predictive preclinical tests for therapeutics that enhance skin repair or limit scarring. Large animals, such as swine, heal in a manner similar to humans, but testing is impractical and expensive. Experiments in mice are more economic, but may be less translatable as this species heals primarily through contraction, not by the processes of epithelialization and granulation tissue formation as seen in human wounds. Here, we describe a murine model of thermal burn injury that closely mimics human healing, resulting in a large, hypertrophic-like scar. This practical, reproducible model is ideal for testing promising wound-healing therapies, such as virus-derived growth factors and immune-modulatory proteins.


Assuntos
Queimaduras/patologia , Cicatriz/prevenção & controle , Modelos Animais de Doenças , Reepitelização/genética , Animais , Queimaduras/genética , Queimaduras/terapia , Cicatriz/genética , Cicatriz/patologia , Feminino , Expressão Gênica , Temperatura Alta , Humanos , Fatores Imunológicos/biossíntese , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Reepitelização/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/lesões , Transgenes , Vírus/genética
15.
ACS Appl Mater Interfaces ; 12(50): 55659-55674, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33327053

RESUMO

Diabetic skin ulcer is one of the severe complications of diabetes mellitus, which has a high incidence and may cause death or disability. Platelet-rich plasma (PRP) is widely used in the treatment of diabetic wounds due to the effect of growth factors (GFs) derived from it. However, the relatively short half-life of GFs limits their applications in clinics. In addition, the presence of a large amount of proteases in the diabetic wound microenvironment results in the degradation of GFs, which further impedes angiogenesis and diabetic wound healing. In our study, we fabricated a self-healing and injectable hydrogel with a composite of chitosan, silk fibroin, and PRP (CBPGCTS-SF@PRP) for promoting diabetic wound healing. CBPGCTS-SF@PRP could protect PRP from enzymatic hydrolysis, release PRP sustainably, and enhance the chemotaxis of mesenchymal stem cells. The results showed that it could promote the proliferation of repair cells in vitro. Moreover, it could enhance wound healing by expediting collagen deposition, angiogenesis, and nerve repair in a type 2 diabetic rat model and a rat skin defect model. We hope that this study will offer a new treatment for diabetic nonhealing wounds in clinics.


Assuntos
Materiais Biocompatíveis/farmacologia , Hidrogéis/química , Plasma Rico em Plaquetas/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Benzaldeídos/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/patologia , Fibroínas/química , Humanos , Hidrogéis/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Fibras Nervosas/fisiologia , Plasma Rico em Plaquetas/química , Polietilenoglicóis/química , Ratos , Regeneração/efeitos dos fármacos , Dermatopatias/tratamento farmacológico , Dermatopatias/patologia
16.
J Med Life ; 13(3): 418-425, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33072218

RESUMO

The study aimed to investigate whether a 3D printed beta-tricalcium phosphate (ß-TCP) scaffold tethered with growth factors and fibrin glue implanted autologous bone marrow-derived mesenchymal stem cells would provide a 3D platform for bone regeneration resulting in new bone formation with plasticity. Twenty 3D printed ß-TCP scaffolds, ten scaffolds engrained with osteogenic mesenchymal stem cells with fibrin glue (group A), and ten scaffolds used as a control group with ß-TCP scaffold and fibrin glue inoculation only (group B) were included in the study. Cell infiltration, migration, and proliferation of human osteogenic stem cells on the scaffolds were executed under both static and dynamic culture conditions. Each scaffold was examined post culture after repeated changes in the nutrient medium at 2, 4 or 8 weeks and assessed for opacity and formation of any bone-like tissues macroscopic, radiographic, and microscopic evaluation. Significant changes in all the prerequisite parameters compiled with an evaluated difference of significance showing maxillofacial skeletal repair via tissue engineering by ß-TCP scaffold and MSCs remains will be the most promising alternative to autologous bone grafts and numerous modalities involving a variety of stem cells, growth factors from platelet-rich fibrin.


Assuntos
Fosfatos de Cálcio/farmacologia , Adesivo Tecidual de Fibrina/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Anormalidades Maxilofaciais/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Impressão Tridimensional , Engenharia Tecidual/métodos , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Humanos , Anormalidades Maxilofaciais/diagnóstico por imagem , Anormalidades Maxilofaciais/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Minerais/análise , Tecidos Suporte/química , Resultado do Tratamento
17.
Int J Nanomedicine ; 15: 6097-6111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884266

RESUMO

The development of biomaterials, stem cells and bioactive factors has led to cartilage tissue engineering becoming a promising tactic to repair cartilage defects. Various polymer three-dimensional scaffolds that provide an extracellular matrix (ECM) mimicking environment play an important role in promoting cartilage regeneration. In addition, numerous growth factors have been found in the regenerative process. However, it has been elucidated that the uncontrolled delivery of these factors cannot fully exert regenerative potential and can also elicit undesired side effects. Considering the complexity of the ECM, neither scaffolds nor growth factors can independently obtain successful outcomes in cartilage tissue engineering. Therefore, collectively, an appropriate combination of growth factors and scaffolds have great potential to promote cartilage repair effectively; this approach has become an area of considerable interest in recent investigations. Of late, an increasing trend was observed in cartilage tissue engineering towards this combination to develop a controlled delivery system that provides adequate physical support for neo-cartilage formation and also enables spatiotemporally delivery of growth factors to precisely and fully exert their chondrogenic potential. This review will discuss the role of polymer scaffolds and various growth factors involved in cartilage tissue engineering. Several growth factor delivery strategies based on the polymer scaffolds will also be discussed, with examples from recent studies highlighting the importance of spatiotemporal strategies for the controlled delivery of single or multiple growth factors in cartilage tissue engineering applications.


Assuntos
Cartilagem , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Polímeros/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Cartilagem/citologia , Cartilagem/fisiologia , Condrogênese , Matriz Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco
18.
Adv Gerontol ; 33(2): 307-312, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32593245

RESUMO

The two-week use of special breathing exercises in combination with the Cortexin peptide bioregulator optimizes the functional state and increases the professional performance of older teachers more effectively compared to the single use of a pharmacological preparation. The maximum effect is noted immediately after the end of the course use of special breathing exercises and the peptide bioregulator and lasts for two months. The results obtained open up prospects for the use of breathing exercises to enhance and prolong the nootropic drugs and antihypoxants antistress and geroprotective effects.


Assuntos
Exercícios Respiratórios , Emprego , Docentes , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Universidades , Idoso , Avaliação Geriátrica , Humanos
19.
Life Sci ; 256: 117964, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534036

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica/efeitos dos fármacos , Cinética , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
PLoS One ; 15(4): e0230265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298282

RESUMO

Autologous adipose tissue (AT) transfer has gained widespread acceptance and is used for a broad variety of regenerative clinical indications. It is assumed that the successful outcome of AT transfer essentially depends on the amount of autocrine-generated growth factors (GF). It is supposed that several GF enhance and improve the anatomic and functional integration of the transplanted AT grafts at the site of implantation. In the present study we have investigated for the first time the correlation between the concentration of GF of freshly isolated AT and the proliferation and migration capacity of mesenchymal stroma cells (MSCs) derived from the respective AT sample. We here show that the proliferation and migration capacity of MSCs strongly depends on the GF content of the AT the cells were isolated from but in an inversely proportional manner. The lower the GF content of an AT sample was, the higher was the proliferation and migration capacity of the respective MSC population contained in the AT and vice versa. Furthermore, we found that supplementation with recombinant GFs only in the case of AT samples with low but not with higher growth factor contents led to a significant enhancement of proliferation and migration of the AT-resident MSCs. As we further show, this inefficiency of GFs to enhance MSC proliferation and migration in AT samples with high GF contents indicates a GF-mediated negative feedback mechanism leading to an impaired GF signaling in MSC obtained from those AT samples. Our results might explain why the successful use of AT grafting is frequently limited by low and unpredictable survival rates, and we suggest to use the knowledge of GF content of harvested AT as a predictive clinical parameter for risk assessment of the therapeutic outcome of autologous AT transfer.


Assuntos
Tecido Adiposo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tecido Adiposo/citologia , Adulto , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade
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