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1.
Mol Cell ; 75(1): 66-75.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31175012

RESUMO

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.


Assuntos
Células Artificiais/metabolismo , Grânulos Citoplasmáticos/genética , Proteínas Intrinsicamente Desordenadas/genética , Peptidomiméticos/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Células Artificiais/citologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Peptidomiméticos/química , Transição de Fase , Plasmídeos/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo
2.
Circ Res ; 134(7): 931-949, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38547250

RESUMO

The ECM (extracellular matrix) is a major component of the vascular microenvironment that modulates vascular homeostasis. ECM proteins include collagens, elastin, noncollagen glycoproteins, and proteoglycans/glycosaminoglycans. ECM proteins form complex matrix structures, such as the basal lamina and collagen and elastin fibers, through direct interactions or lysyl oxidase-mediated cross-linking. Moreover, ECM proteins directly interact with cell surface receptors or extracellular secreted molecules, exerting matricellular and matricrine modulation, respectively. In addition, extracellular proteases degrade or cleave matrix proteins, thereby contributing to ECM turnover. These interactions constitute the ECM interactome network, which is essential for maintaining vascular homeostasis and preventing pathological vascular remodeling. The current review mainly focuses on endogenous matrix proteins in blood vessels and discusses the interaction of these matrix proteins with other ECM proteins, cell surface receptors, cytokines, complement and coagulation factors, and their potential roles in maintaining vascular homeostasis and preventing pathological remodeling.


Assuntos
Proteínas da Matriz Extracelular , Matriz Extracelular , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Colágeno/metabolismo , Elastina/metabolismo , Homeostase , Receptores de Superfície Celular/metabolismo
3.
J Immunol ; 213(1): 75-85, 2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38758115

RESUMO

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.


Assuntos
Elastina , Neutrófilos , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Proteólise , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Neutrófilos/imunologia , Elastina/metabolismo , Feminino , Masculino , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Pessoa de Meia-Idade , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Enfisema Pulmonar/imunologia , Idoso , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Citrulinação , Desiminases de Arginina em Proteínas/metabolismo , Elastase de Leucócito/metabolismo , Pulmão/imunologia , Pulmão/patologia
4.
Am J Hum Genet ; 109(12): 2230-2252, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36351433

RESUMO

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.


Assuntos
Doenças Ósseas Metabólicas , Cútis Laxa , Animais , Humanos , Camundongos , Colágeno/genética , Cútis Laxa/genética , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo
5.
Circ Res ; 132(1): 72-86, 2023 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-36453283

RESUMO

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.


Assuntos
Infarto do Miocárdio , Miocárdio , Humanos , Ratos , Animais , Miocárdio/metabolismo , Elastina/metabolismo , Tropoelastina/genética , Tropoelastina/metabolismo , Cicatriz , Volume Sistólico , Função Ventricular Esquerda , Miócitos Cardíacos/metabolismo , Colágeno/metabolismo , Remodelação Ventricular
6.
Arterioscler Thromb Vasc Biol ; 44(7): 1674-1682, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38752350

RESUMO

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln+/- mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln+/- mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln+/- mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.


Assuntos
Estenose Aórtica Supravalvular , Catequina , Proliferação de Células , Modelos Animais de Doenças , Elastina , Células-Tronco Pluripotentes Induzidas , Músculo Liso Vascular , Miócitos de Músculo Liso , Elastina/metabolismo , Animais , Humanos , Catequina/análogos & derivados , Catequina/farmacologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Estenose Aórtica Supravalvular/metabolismo , Estenose Aórtica Supravalvular/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Células Cultivadas , Camundongos Endogâmicos C57BL , Feminino , Masculino , Camundongos Knockout
7.
Am J Physiol Cell Physiol ; 327(3): C698-C715, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38946422

RESUMO

Type 2 diabetes (T2D) constitutes a major public health problem, and despite prevention efforts, this pandemic disease is one of the deadliest diseases in the world. In 2022, 6.7 million patients with T2D died prematurely from vascular complications. Indeed, diabetes increases the risk of myocardial infarction or stroke eightfold. The identification of the molecular factors involved in the occurrence of cardiovascular complications and their prevention are therefore major axes. Our hypothesis is that factors brought into play during physiological aging appear prematurely with diabetes progression. Our study focused on the aging of the extracellular matrix (ECM), a major element in the maintenance of vascular homeostasis. We characterized the morphological and functional aspects of aorta, with a focus on the collagen and elastic fibers of diabetic mice aged from 6 mo to nondiabetic mice aged 6 mo and 20 mo. The comparison with the two nondiabetic models (young and old) highlighted an exacerbated activity of proteases, which could explain a disturbance in the collagen accumulation and an excessive degradation of elastic fibers. Moreover, the generation of circulating elastin-derived peptides reflects premature aging of the ECM. These extracellular elements contribute to the appearance of vascular rigidity, often the origin of pathologies such as hypertension and atherosclerosis. In conclusion, we show that diabetic mice aged 6 mo present the same characteristics of ECM wear as those observed in mice aged 20 mo. This accelerated aortic wall remodeling could then explain the early onset of cardiovascular diseases and, therefore, the premature death of patients with T2D.NEW & NOTEWORTHY Aortic elastic fibers of young (6-mo old) individuals with diabetes degrade prematurely and exhibit an appearance like that found in aged (20-mo old) nondiabetic mice. Exacerbated elastolysis and elastin-derived peptide production are characteristic elements, contributing to early aortic wall rigidity and hypertension development. Therefore, limiting this early aging could be a judicious therapeutic approach to reduce cardiovascular complications and premature death in patients with diabetes.


Assuntos
Aorta , Tecido Elástico , Matriz Extracelular , Síndrome Metabólica , Camundongos Endogâmicos C57BL , Rigidez Vascular , Animais , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Rigidez Vascular/fisiologia , Camundongos , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Elastina/metabolismo , Colágeno/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Envelhecimento/patologia , Envelhecimento/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Senilidade Prematura/metabolismo , Senilidade Prematura/patologia , Senilidade Prematura/fisiopatologia
8.
Am J Physiol Lung Cell Mol Physiol ; 326(6): L812-L820, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38712445

RESUMO

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and obstruction, primarily caused by tobacco smoking. Although the involvement of immune cells in COPD pathogenesis is well established, the contribution of innate lymphoid cells (ILCs) remains poorly understood. ILCs are a type of innate immune cells that participate in tissue remodeling processes, but their specific role in COPD has not been fully elucidated. During COPD, the breakdown of pulmonary elastin generates elastin peptides that elicit biological activities on immune cells. This study aimed to investigate the presence of ILC in patients with COPD and examine the impact of elastin peptides on their functionality. Our findings revealed an elevated proportion of ILC2 in the peripheral blood of patients with COPD, and a general activation of ILC as indicated by an increase in their cytokine secretion capacity. Notably, our study demonstrated that serum from patients with COPD promotes ILC2 phenotype, likely due to the elevated concentration of IL-5, a cytokine known to favor ILC2 activation. Furthermore, we uncovered that this increase in IL-5 secretion is partially attributed to its secretion by macrophages upon stimulation by elastin peptides, suggesting an indirect role of elastin peptides on ILC in COPD. These findings shed light on the involvement of ILC in COPD and provide insights into the potential interplay between elastin breakdown, immune cells, and disease progression. Further understanding of the mechanisms underlying ILC activation and their interaction with elastin peptides could contribute to the development of novel therapeutic strategies for COPD management.NEW & NOTEWORTHY Elastin-derived peptides, generated following alveolar degradation during emphysema in patients with COPD, are able to influence the response of type 2 innate lymphoid cells. We show that the orientation of innate lymphoid cells in patients with COPD is shifted toward a type 2 profile and that elastin peptides are indirectly participating in that shift through their influence of macrophages, which in turn impact innate lymphoid cells.


Assuntos
Elastina , Imunidade Inata , Linfócitos , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Elastina/metabolismo , Elastina/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/efeitos dos fármacos , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Interleucina-5/metabolismo , Interleucina-5/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Peptídeos/farmacologia , Peptídeos/imunologia
9.
Am J Physiol Lung Cell Mol Physiol ; 327(3): L304-L318, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38915286

RESUMO

Extracellular matrix (ECM) remodeling has been implicated in the irreversible obstruction of airways and destruction of alveolar tissue in chronic obstructive pulmonary disease (COPD). Studies investigating differences in the lung ECM in COPD have mainly focused on some collagens and elastin, leaving an array of ECM components unexplored. We investigated the differences in the ECM landscape comparing severe-early onset (SEO)-COPD and moderate COPD to control lung tissue for collagen type I α chain 1 (COL1A1), collagen type VI α chain 1 (COL6A1); collagen type VI α chain 2 (COL6A2), collagen type XIV α chain 1 (COL14A1), fibulin 2 and 5 (FBLN2 and FBLN5), latent transforming growth factor ß binding protein 4 (LTBP4), lumican (LUM), versican (VCAN), decorin (DCN), and elastin (ELN) using image analysis and statistical modeling. Percentage area and/or mean intensity of expression of LUM in the parenchyma, and COL1A1, FBLN2, LTBP4, DCN, and VCAN in the airway walls, was proportionally lower in COPD compared to controls. Lowered levels of most ECM proteins were associated with decreasing forced expiratory volume in 1 s (FEV1) measurements, indicating a relationship with disease severity. Furthermore, we identified six unique ECM signatures where LUM and COL6A1 in parenchyma and COL1A1, FBLN5, DCN, and VCAN in airway walls appear essential in reflecting the presence and severity of COPD. These signatures emphasize the need to examine groups of proteins to represent an overall difference in the ECM landscape in COPD that are more likely to be related to functional effects than individual proteins. Our study revealed differences in the lung ECM landscape between control and COPD and between SEO and moderate COPD signifying distinct pathological processes in the different subgroups.NEW & NOTEWORTHY Our study identified chronic obstructive pulmonary disease (COPD)-associated differences in the lung extracellular matrix (ECM) composition. We highlight the compartmental differences in the ECM landscape in different subtypes of COPD. The most prominent differences were observed for severe-early onset COPD. Moreover, we identified unique ECM signatures that describe airway walls and parenchyma providing insight into the intertwined nature and complexity of ECM changes in COPD that together drive ECM remodeling and may contribute to disease pathogenesis.


Assuntos
Decorina , Elastina , Proteínas da Matriz Extracelular , Matriz Extracelular , Pulmão , Doença Pulmonar Obstrutiva Crônica , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pulmão/metabolismo , Pulmão/patologia , Feminino , Proteínas da Matriz Extracelular/metabolismo , Elastina/metabolismo , Decorina/metabolismo , Idoso , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Versicanas/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Proteínas de Ligação a TGF-beta Latente/genética , Lumicana/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Índice de Gravidade de Doença , Colágeno Tipo VI/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 327(4): L600-L606, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39137524

RESUMO

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. This study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.NEW & NOTEWORTHY This study provides evidence for the impact of peptidyl arginine deiminase (PAD) enzymes on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.


Assuntos
Citrulinação , Elastina , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica , Animais , Masculino , Camundongos , alfa 1-Antitripsina/metabolismo , Citrulina/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/patologia
11.
J Neurochem ; 168(8): 1460-1474, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38168728

RESUMO

Extracellular elastin-derived peptides (EDPs) accumulate in the aging brain and have been associated with vascular dementia and Alzheimer's disease (AD). The activation of inflammatory processes in glial cells with EDP treatment has received attention, but not in neurons. To properly understand EDPs' pathogenic significance, the impact on neuronal function and neuron-microglia crosstalk was explored further. Among the EDP molecules, Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a typical repeating hexapeptide. Here, we observed that EDPs-VGVAPG influenced neuronal survival and morphology in a dose-dependent manner. High concentrations of VGVAPG induced synapse loss and microglia hyperactivation in vivo and in vitro. Following EDP incubation, galectin 3 (Gal-3) released by neurons served as a chemokine, attracting microglial engulfment. Blocking Gal-3 and EDP binding remedied synapse loss in neurons and phagocytosis in microglia. In response to the accumulation of EDPs, proteomics in matrix remodeling and cytoskeleton dynamics, such as a disintegrin and metalloproteinase (ADAM) family, were engaged. These findings in extracellular EDPs provided more evidence for the relationship between aging and neuron dysfunction, increasing the insight of neuroinflammatory responses and the development of new specialized extracellular matrix remolding-targeted therapy options for dementia or other neurodegenerative disease.


Assuntos
Envelhecimento , Encéfalo , Elastina , Microglia , Neurônios , Neurônios/metabolismo , Neurônios/patologia , Animais , Elastina/metabolismo , Microglia/metabolismo , Microglia/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Oligopeptídeos/farmacologia , Oligopeptídeos/metabolismo , Camundongos , Masculino , Comunicação Celular/fisiologia , Camundongos Endogâmicos C57BL , Células Cultivadas , Galectina 3/metabolismo , Humanos
12.
Cytokine ; 182: 156725, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39106575

RESUMO

During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (ß-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells.


Assuntos
Diferenciação Celular , Elastina , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Elastina/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Antígeno Ki-67/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Peptídeos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Células Cultivadas , Oligopeptídeos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proliferação de Células , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo
13.
Exp Dermatol ; 33(3): e15052, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38483134

RESUMO

Skin forms the outer barrier of the body. Upon injury, successful wound healing in normal skin restores tissue damage and counteracts the loss of extracellular matrix (ECM) proteins and cells. Collagens and elastin are the most abundant structural proteins of the ECM. In homeostasis, collagen type I is the prevalent form, but it is replaced by type III collagen upon wounding, and only later remodelled. In turn, unsuccessful healing results in scars, which tend to be inflexible and inelastic as compared to normal elastic dermis. Scar inelasticity may be due to the absence of mature elastin fibre formation and cross-linking. In this review, the available information on the process of formation of new collagen and elastic fibres during wound healing is analysed. The distinct roles of elastin and collagen proteins during healing are revisited and future research directions proposed which may help improve clinical management of open wounds and scars.


Assuntos
Cicatriz , Matriz Extracelular , Humanos , Cicatriz/metabolismo , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Pele/metabolismo , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Cicatrização
14.
FASEB J ; 37(8): e23077, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37402128

RESUMO

Inflammatory processes are activated following ischemic stroke that lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to the drug carrier elastin-like polypeptide (ELP), decreases NF-κB induced inflammatory cytokine production in cultured macrophages, crosses the plasma membrane and accumulates in the cytoplasm of both neurons and microglia in vitro, and accumulates at the infarct site where the blood-brain barrier (BBB) is compromised following middle cerebral artery occlusion (MCAO) in rats. Additionally, SynB1-ELP-p50i treatment reduces infarct volume by 11.86% compared to saline-treated controls 24 h following MCAO. Longitudinally, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Ratos , Animais , NF-kappa B/metabolismo , AVC Isquêmico/metabolismo , Elastina/metabolismo , Encéfalo/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Acidente Vascular Cerebral/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Microglia/metabolismo
15.
Transgenic Res ; 33(1-2): 21-33, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38573429

RESUMO

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.


Assuntos
Polipeptídeos Semelhantes à Elastina , Seda , Seda/genética , Proteínas de Artrópodes , Elastina/genética , Elastina/química , Elastina/metabolismo , Nicotiana/genética , Proteínas Recombinantes de Fusão/genética
16.
Protein Expr Purif ; 222: 106521, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38852714

RESUMO

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/µg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/µg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.


Assuntos
Proteínas de Bactérias , Elastina , Nicotiana , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão , Shigella dysenteriae , Nicotiana/genética , Nicotiana/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Elastina/genética , Elastina/química , Elastina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Shigella dysenteriae/genética , Medicago sativa/genética , Medicago sativa/metabolismo , Medicago sativa/química , Medicago sativa/microbiologia , Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Glicoproteínas/genética , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Polipeptídeos Semelhantes à Elastina
17.
Circ Res ; 130(5): 728-740, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35135328

RESUMO

BACKGROUND: Marfan syndrome (MFS) is associated with TGF (transforming growth factor) ß-stimulated ERK (extracellular signal-regulated kinase) activity in vascular smooth muscle cells (VSMCs), which adopt a mixed synthetic/contractile phenotype. In VSMCs, TGFß induces IL (interleukin) 11) that stimulates ERK-dependent secretion of collagens and MMPs (matrix metalloproteinases). Here, we examined the role of IL11 in the MFS aorta. METHODS: We used echocardiography, histology, immunostaining, and biochemical methods to study aortic anatomy, physiology, and molecular endophenotypes in Fbn1C1041G/+ mice, an established murine model of MFS (mMFS). mMFS mice were crossed to an IL11-tagged EGFP (enhanced green fluorescent protein; Il11EGFP/+) reporter strain or to a strain deleted for the IL11 receptor (Il11ra1-/-). In therapeutic studies, mMFS were administered an X209 (neutralizing antibody against IL11RA [IL11 receptor subunit alpha]) or IgG for 20 weeks and imaged longitudinally. RESULTS: IL11 mRNA and protein were elevated in the aortas of mMFS mice, as compared to controls. mMFS mice crossed to Il11EGFP/+ mice had increased IL11 expression in VSMCs, notably in the aortic root and ascending aorta. As compared to the mMFS parental strain, double mutant mMFS:Il11ra1-/- mice had reduced aortic dilatation and exhibited lesser fibrosis, inflammation, elastin breaks, and VSMC loss, which was associated with reduced aortic COL1A1 (collagen type I alpha 1 chain), IL11, MMP2/9, and phospho-ERK expression. To explore therapeutic targeting of IL11 signaling in MFS, we administered either a neutralizing antibody against IL11RA (X209) or an IgG control. After 20 weeks of antibody administration, as compared to IgG, mMFS mice receiving X209 had reduced thoracic and abdominal aortic dilation as well as lesser fibrosis, inflammation, elastin breaks, and VSMC loss. By immunoblotting, X209 was shown to reduce aortic COL1A1, IL11, MMP2/9, and phospho-ERK expression. CONCLUSIONS: In MFS, IL11 is upregulated in aortic VSMCs to cause ERK-related thoracic aortic dilatation, inflammation, and fibrosis. Therapeutic inhibition of IL11, imminent in clinical trials, might be considered as a new approach in MFS.


Assuntos
Doenças da Aorta , Síndrome de Marfan , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Aorta/metabolismo , Doenças da Aorta/patologia , Modelos Animais de Doenças , Elastina/metabolismo , Fibrose , Imunoglobulina G/metabolismo , Inflamação/metabolismo , Interleucina-11/metabolismo , Subunidade alfa de Receptor de Interleucina-11 , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , Receptores de Interleucina-11/metabolismo , Fator de Crescimento Transformador beta/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 43(12): 2301-2311, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37855127

RESUMO

BACKGROUND: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in nonhuman primates. METHODS: Aortic samples were harvested from the ascending, descending thoracic, suprarenal, and infrarenal regions of young control monkeys and adult monkeys with high fructose consumption for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses, respectively. RESULTS: Immunostaining of CD31 and αSMA (alpha-smooth muscle actin) revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared with other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared with other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys with high fructose consumption displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. CONCLUSIONS: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3.


Assuntos
Aorta Abdominal , Tecido Elástico , Animais , Camundongos , Aorta Abdominal/metabolismo , Macaca fascicularis/metabolismo , Tecido Elástico/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Elastina/metabolismo , Colágeno/metabolismo , Frutose
19.
Arterioscler Thromb Vasc Biol ; 43(6): e210-e217, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37021575

RESUMO

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially lethal disease that lacks pharmacological treatment. Degradation of extracellular matrix proteins, especially elastin laminae, is the hallmark for AAA development. DOCK2 (dedicator of cytokinesis 2) has shown proinflammatory effects in several inflammatory diseases and acts as a novel mediator for vascular remodeling. However, the role of DOCK2 in AAA formation remains unknown. METHODS: Ang II (angiotensin II) infusion of ApoE-/- (apolipoprotein E deficient) mouse and topical elastase-induced AAA combined with DOCK2-/- (DOCK2 knockout) mouse models were used to study DOCK2 function in AAA formation/dissection. The relevance of DOCK2 to human AAA was examined using human aneurysm specimens. Elastin fragmentation in AAA lesion was observed by elastin staining. Elastin-degrading enzyme MMP (matrix metalloproteinase) activity was measured by in situ zymography. RESULTS: DOCK2 was robustly upregulated in AAA lesion of Ang II-infused ApoE-/- mice, elastase-treated mice, as well as human AAA lesions. DOCK2-/- significantly attenuated the Ang II-induced AAA formation/dissection or rupture in mice along with reduction of MCP-1 (monocyte chemoattractant protein-1) and MMP expression and activity. Accordingly, the elastin fragmentation observed in ApoE-/- mouse aorta infused with Ang II and elastase-treated aorta was significantly attenuated by DOCK2 deficiency. Moreover, DOCK2-/- decreased the prevalence and severity of aneurysm formation, as well as the elastin degradation observed in the topical elastase model. CONCLUSIONS: Our results indicate that DOCK2 is a novel regulator for AAA formation. DOCK2 regulates AAA development by promoting MCP-1 and MMP2 expression to incite vascular inflammation and elastin degradation.


Assuntos
Aneurisma da Aorta Abdominal , Elastina , Humanos , Animais , Camundongos , Elastina/metabolismo , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/prevenção & controle , Camundongos Knockout , Apolipoproteínas E , Elastase Pancreática/farmacologia , Angiotensina II/farmacologia , Modelos Animais de Doenças , Aorta Abdominal/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Ativadoras de GTPase/metabolismo
20.
Mol Cell Neurosci ; 127: 103902, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37918553

RESUMO

Autophagy is a self-degradative process important for balancing the sources of energy and involved in the development of Alzheimer's disease (AD). To date, a number of papers have shown that elastin-derived peptides (EDPs) affect the expression and activation of peroxisome proliferator-activated receptor gamma (PPARγ), which is crucial for the development of AD and autophagy initiation. Therefore, the aim of the present study was to determine whether EDPs with a Val-Gly-Val-Ala-Pro-Gly (VGVAPG) amino acid sequence activate the autophagic process in undifferentiated SH-SY5Y human neuroblastoma cells. Our study is the first to show that EDPs with the VGVAPG sequence initiate the autophagy process in the undifferentiated SH-SY5Y cell line exhibiting a number of features of normal neuroblasts. In particular, we observed in our study that VGAVPG peptide increased ULK1, AKT, PPARγ, and LC3B protein expression. Moreover, our experiments with the agonist (rosiglitazone) and antagonist (GW9662) of PPARγ confirm that the studied EDP acts through the PPARγ pathway affecting mTOR and finally autophagy. Some studies have shown that autophagy disturbances are involved in the development of AD. Therefore, we believe that our study will provide new evidence of the possible involvement of EDPs (especially VGVAPG) in the development of AD.


Assuntos
Neuroblastoma , PPAR gama , Humanos , PPAR gama/metabolismo , Elastina/química , Elastina/metabolismo , Peptídeos , Autofagia
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