RESUMO
The synthesis and assembly of mature, organized elastic fibers remains a limitation to the clinical use of many engineered tissue replacements. There is a critical need for a more in-depth understanding of elastogenesis regulation for the advancement of methods to induce and guide production of elastic matrix structures in engineered tissues that meet the structural and functional requirements of native tissue. The dramatic increase in elastic fibers through normal pregnancy has led us to explore the potential role of mechanical stretch in combination with pregnancy levels of the steroid hormones 17ß-estradiol and progesterone on elastic fiber production by human uterine myometrial smooth muscle cells in a three-dimensional (3D) culture model. Opposed to a single strain regimen, we sought to better understand how the amplitude and frequency parameters of cyclic strain influence elastic fiber production in these myometrial tissue constructs (MTC). Mechanical stretch was applied to MTC at a range of strain amplitudes (5%, 10%, and 15% at 0.5 Hz frequency) and frequencies (0.1 Hz, 0.5 Hz, 1 Hz, and constant 0 Hz at 10% amplitude), with and without pregnancy-level hormones, for 6 days. MTC were assessed for cell proliferation, matrix elastin protein content, and expression of the main elastic fiber genes, tropoelastin (ELN) and fibrillin-1 (FBN1). Significant increases in elastin protein and ELN and FBN1 mRNA were produced from samples subjected to a 0.5 Hz, 10% strain regimen, as well as samples stretched at higher amplitude (15%, 0.5 Hz) and higher frequency (1 Hz, 10%); however, no significant effects because of third-trimester mimetic hormone treatment were determined. These results establish that a minimum level of strain is required to stimulate the synthesis of elastic fiber components in our culture model and show this response can be similarly enhanced by increasing either the amplitude or frequency parameter of applied strain. Further, our results demonstrate strain alone is sufficient to stimulate elastic fiber production and suggest hormones may not be a significant factor in regulating elastin synthesis. This 3D culture model will provide a useful tool to further investigate mechanisms underlying pregnancy-induced de novo elastic fiber synthesis and assembly by uterine smooth muscle cells.
Assuntos
Elastina , Miométrio , Estresse Mecânico , Humanos , Feminino , Elastina/metabolismo , Elastina/biossíntese , Miométrio/metabolismo , Miométrio/citologia , Células Cultivadas , Gravidez , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Tropoelastina/metabolismo , Técnicas de Cultura de Células em Três Dimensões/métodos , Fibrilina-1/metabolismo , Fibrilinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Engenharia Tecidual/métodos , Estradiol/biossíntese , Estradiol/farmacologia , Estradiol/metabolismo , Modelos Biológicos , AdipocinasRESUMO
Vascular mural cells (vMCs) play an essential role in the development and maturation of the vasculature by promoting vessel stabilization through their interactions with endothelial cells. Whether endothelial metabolism influences mural cell recruitment and differentiation is unknown. Here, we show that the oxidative pentose phosphate pathway (oxPPP) in endothelial cells is required for establishing vMC coverage of the dorsal aorta during early vertebrate development in zebrafish and mice. We demonstrate that laminar shear stress and blood flow maintain oxPPP activity, which in turn, promotes elastin expression in blood vessels through production of ribose-5-phosphate. Elastin is both necessary and sufficient to drive vMC recruitment and maintenance when the oxPPP is active. In summary, our work demonstrates that endothelial cell metabolism regulates blood vessel maturation by controlling vascular matrix composition and vMC recruitment.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Matriz Extracelular/metabolismo , Fosforilação Oxidativa , Via de Pentose Fosfato , Animais , Biomarcadores , Elastina/biossíntese , Elastina/genética , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Expressão Gênica , Genes Reporter , Glucose/metabolismo , Hemodinâmica , Camundongos , Camundongos Knockout , Modelos Biológicos , Estresse Oxidativo , Pentosefosfatos/metabolismo , Peixe-ZebraRESUMO
Lack of adult cells' ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.
Assuntos
Monofosfato de Adenosina/farmacologia , Derme/fisiologia , Elastina/biossíntese , Estradiol/farmacologia , Caracteres Sexuais , Pele Artificial , Engenharia Tecidual , Adulto , Meios de Cultura , AMP Cíclico/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto JovemRESUMO
Reduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.
Assuntos
Derme/metabolismo , Elastina/biossíntese , Neuraminidase , Administração Cutânea , Animais , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Masculino , Neuraminidase/metabolismo , Neuraminidase/farmacologia , Ratos , Ratos WistarRESUMO
We investigated the bladder and urethral function in a rat model lacking the protein lysyl oxidase-like 1 (Loxl1). Female nulliparous rats of Loxl1-/- or age-matched wild type (WT) rats had leak-point pressure testing, cystometry, histopathological analyses of lower urinary tract, and contractile response of isolated detrusor strips to carbachol and electric field stimulation. The Loxl1-/- rats showed increased looseness and redundancy of the skin, the decreased intercontraction interval and voided volume in cystometry, the lower leak-point pressure, thinner elastic fibers of the mesentery, bladder, urethra and vagina, and smaller contractile response of detrusor strips to carbachol when compared to the WT rats. Thus, the insufficient hydrostatic mechanism of urethra via submucosal impaired elastin synthesis might reduce the resting urethral closure pressure and the diminished cholinergic contractile response of detrusor smooth muscle might be involved in bladder activity in the Loxl1-/- rats.
Assuntos
Aminoácido Oxirredutases/biossíntese , Elastina/biossíntese , Uretra/fisiopatologia , Aminoácido Oxirredutases/genética , Animais , Tecido Elástico/metabolismo , Estimulação Elétrica , Feminino , Genótipo , Contração Muscular , Músculo Liso/metabolismo , Pressão , Ratos , Ratos Sprague-Dawley , Resistência à Tração , Uretra/metabolismo , Bexiga Urinária/fisiopatologia , Sistema UrinárioRESUMO
Skin aging appears to be the result of overlapping intrinsic (including genetic and hormonal factors) and extrinsic (external environment including chronic light exposure, chemicals, and toxins) processes. These factors cause decreases in the synthesis of collagen type I and elastin in fibroblasts and increases in the melanin in melanocytes. Collagen Type I is the most abundant type of collagen and is a major structural protein in human body tissues. In previous studies, many products containing collagen derived from land and marine animals as well as other sources have been used for a wide range of purposes in cosmetics and food. However, to our knowledge, the effects of human collagenderived peptides on improvements in skin condition have not been investigated. Here we isolate and identify the domain of a human COL1A2-derived protein which promotes fibroblast cell proliferation and collagen type I synthesis. This human COL 1A2-derived peptide enhances wound healing and elastin production. Finally, the human collagen alpha-2 type I-derived peptide (SMM) ameliorates collagen type I synthesis, cell proliferation, cell migration, and elastin synthesis, supporting a significant anti-wrinkle effect. Collectively, these results demonstrate that human collagen alpha-2 type I-derived peptides is practically accessible in both cosmetics and food, with the goal of improving skin condition. [BMB Reports 2020; 53(10): 539-544].
Assuntos
Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Pele/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Colágeno/metabolismo , Colágeno Tipo I/fisiologia , Elastina/biossíntese , Elastina/metabolismo , Elastina/farmacologia , Humanos , Envelhecimento da Pele/fisiologia , Cicatrização/fisiologiaRESUMO
This work unveils a fish collagen drink for improvement of skin aging. Previous studies frequently discussed the skin aging from the angle of the representative characteristics of collagen loss and the oxidative-induced expression of proteolytic enzymes matrix metalloproteinases (MMPs), but few groups comprehensively investigated the efficacy of oral hydrolyzed collagen for enhancing protein folding and DNA repair as well as improving notable cell behaviors. To delineate the broad perspective on delaying skin aging, we inspected the collagen drink-treated fibroblast cells from the molecular and cellular aspects. The results show that the collagen drink could perform the compact antiaging effects on ROS inhibition, the facilitation of the synthesis of extracellular matrix (ECM) proteins, the increase of mitochondrial activity, and improvement of the gene expression regarding correct protein folding, DNA mismatch repair (MMR) and base excision repair (BER). Although the experimental results are built on the cellular models, we believe that the positive outcomes can provide more details on the influence of oral hydrolyzed collagen supplement for antiaging. In short, we have successfully proved that the synergistic effect of the collagen drink could not only reduce the oxidative damage but also ameliorate the cell functionality to compensate the harmful effects induced by UVA.
Assuntos
Antioxidantes/farmacologia , Bebidas , Senescência Celular/efeitos dos fármacos , Colágeno/administração & dosagem , Colágeno/biossíntese , Reparo do DNA , Fibroblastos/metabolismo , Dobramento de Proteína , Administração Oral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Elastina/biossíntese , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Dobramento de Proteína/efeitos dos fármacos , Dobramento de Proteína/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Raios UltravioletaRESUMO
Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.
Assuntos
Proteínas de Bactérias , Celulase , Elastina , Proteínas Recombinantes de Fusão , Thermotoga maritima/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Celulase/biossíntese , Celulase/química , Celulase/genética , Celulase/isolamento & purificação , Elastina/biossíntese , Elastina/química , Elastina/genética , Elastina/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Thermotoga maritima/enzimologiaRESUMO
Ionizable amino acids in protein-based hydrogels can confer pH-responsive behavior. Because elastin-like polypeptides (ELPs) have an established sequence and can crosslink to form hydrogels, they are an ideal system for creating pH-sensitive materials. This study examines different parameters that might affect pH-sensitive behavior and characterizes the mechanical and physical properties between pH 3 and 11 of three ELP-based crosslinked hydrogels. The first finding is that varying the amount of crosslinker affects the overall stiffness and resilience of the hydrogels but does not strongly affect water content, swelling ratio, or pH sensitivity. Second, the choice of two popular tag sequences, which vary in histidine and aspartic acid content, does not have a strong effect on pH-sensitive properties. Last, selectively blocking lysine and tyrosine residues through acetylation significantly decreases the pH-sensitive zeta potential. Acetylated hydrogels also demonstrate different behavior at low pH values with reduced swelling, reduced water content, and higher stiffness. Overall, this work demonstrates that ELP hydrogels with ionizable groups are promising materials for environmentally-responsive applications such as drug delivery, tissue engineering, and microfluidics.
Assuntos
Materiais Biocompatíveis/química , Elastina/química , Hidrogéis/química , Engenharia de Proteínas/métodos , Água/química , Acetilação , Sequência de Aminoácidos , Ácido Aspártico/química , Sistemas de Liberação de Medicamentos , Elastina/biossíntese , Elastina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Dispositivos Lab-On-A-Chip , Lisina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Engenharia Tecidual/métodos , Tirosina/química , MolhabilidadeRESUMO
BACKGROUND: Structural changes in collagen and elastin fiber density have been previously evaluated by qualitative histological studies; however, quantitative evaluations are lacking. AIM: To evaluate quantitative changes in collagen and elastin fibers in the vaginal wall in a porcine model after volumetric radiofrequency heating with an intravaginal applicator. METHODS: An animal model was used (domestic pig, multipara: 5.67 ± 0.94 deliveries, 3 years of age). Three pigs under general anesthesia were treated (8-minute, vaginal canal area) once per week for the course of three weeks. There were 2 follow-up evaluations at one and four weeks. Histology specimens were obtained via punch biopsy under ultrasound control. Ultrasound video measurements of the vaginal wall thickness were also obtained. Tissue samples were stained by H&E as well as stains for collagen and elastin fibers. RESULTS: Elastin (P < .001) and collagen (P < .01) fiber density increased after every treatment. The measured increase in fibers was highest at the one-week follow-up. Elastin accounted on average for 51.46 ± 16.86% of the tissue examined (increase of 36.8% points), while collagen accounted on average for 44.83 ± 18.92% (increase of 17.1% points). The number of synthetically active cells was increased by 16%. While vaginal wall thickness did show an increase of 1.66 mm (32%), this tendency was not statistically significant (P > .05). CONCLUSION: Results suggest that volumetric heating of vaginal tissue produced quantitative improvement in the connective tissue organization in a porcine study. Neocollagenesis and neoelastogenesis were observed with an increased number of synthetically active cells.
Assuntos
Colágeno , Elastina , Terapia por Radiofrequência , Vagina , Doenças Vaginais , Animais , Feminino , Colágeno/análise , Colágeno/biossíntese , Tecido Conjuntivo/patologia , Tecido Conjuntivo/efeitos da radiação , Modelos Animais de Doenças , Elastina/análise , Elastina/biossíntese , Terapia por Radiofrequência/instrumentação , Terapia por Radiofrequência/métodos , Sus scrofa , Vagina/patologia , Vagina/efeitos da radiação , Doenças Vaginais/patologia , Doenças Vaginais/terapiaRESUMO
Elastin-like polymers (ELPs) are frequently used in a variety of bioengineering applications because of their stimuli-responsive properties. Above their transition temperature, ELPs will adopt different structures that promote intra- and intermolecular hydrophobic contacts to minimize unfavorable interactions with an aqueous environment. We electrochemically characterize the stimuli-responsive behavior of surface-immobilized ELPs corresponding to two proposed states: extended and collapsed. In the extended state the ELPs are more solvated. In the collapsed state, triggered by introducing an environmental stimulus, non-polar intramolecular contacts within ELPs are favored, resulting in quantifiable morphological changes on the surface characterized using electrochemical impedance spectroscopy (EIS). Charge transfer resistance, a component of impedance, was shown to increase after exposing an ELP modified electrode to a high salt concentration environment (3.0 M NaCl). An increase in charge transfer resistance indicates an increase in the insulating layer on the electrode surface consistent with the proposed mechanism of collapse, as the ELPs have undergone morphological changes to hinder the kinetics of the redox couple exchange. Further characterization of the surface-immobilized ELPs showed a reproducible surface modification, as well as reversibility and tunability of the stimuli-response.
Assuntos
Elastina/química , Cloreto de Sódio/química , Espectroscopia Dielétrica , Elastina/biossíntese , Técnicas Eletroquímicas , Escherichia coli/genética , Escherichia coli/metabolismo , Ouro/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.
Assuntos
Sistema Livre de Células , Escherichia coli/metabolismo , Biossíntese de Proteínas , Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Elastina/biossíntese , Proteínas de Escherichia coli/genética , Fatores de Terminação de Peptídeos/deficiência , Fatores de Terminação de Peptídeos/genética , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Previous studies have shown that calcium hydroxylapatite (CaHa) injected intradermally resulted in new collagen production at 6 months after injection, and a possible increase in elastin formation. In addition, a recent study showed the formation of new collagen, elastin, and angiogenesis after injection at 4 and 9 months. OBJECTIVE: To evaluate any changes in the presence of elastic fibers, proteoglycans, and elastin in photodamaged skin after injections with CaHa. METHODS AND MATERIALS: Fifteen subjects underwent a punch biopsy of the right infra-auricular areas before and after injection of CaHa on day 180. Specimens were stained for elastic fibers, elastin, and proteoglycan presence. RESULTS: Quantitative analysis demonstrated a percent change in elastic fibers varying between 29% and 179% at 6 months in comparison with baseline. Subjects showed an increase in elastin between 12% and 66%. Subjects had positive mean percentage change in proteoglycans of 76.27% (t-test of 0.198). CONCLUSION: This is the first study to show that CaHa can increase proteoglycans and echoed previous studies showing it can also have an effect on elastin, which indicates it can induce remodeling of all aspects of the extracellular matrix. Much larger and longer studies are required to confirm its unique impact on collagen, elastin, and proteoglycans.
Assuntos
Materiais Biocompatíveis/farmacologia , Durapatita/farmacologia , Tecido Elástico/patologia , Elastina/biossíntese , Proteoglicanas/biossíntese , Envelhecimento da Pele/efeitos dos fármacos , Materiais Biocompatíveis/administração & dosagem , Biópsia , Colágeno/biossíntese , Durapatita/administração & dosagem , Tecido Elástico/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Injeções Intradérmicas , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Envelhecimento da Pele/fisiologiaRESUMO
Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25 µmol/L and 100 µmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0 mol/L NaCl at 32 °C for 10 min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30 °C, 7.5 and 30 min, respectively. The enzyme activity was maintained at over 50% at 45 °C for 12 h. The enzyme specific activity was 438.8 ± 2.1 U/µmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe2+, and Cu2+ inhibited enzyme activity, and Mg2+, Zn2+, Ca2+, and K+ had no significant effects on it.
Assuntos
Arsênio/metabolismo , Elastina/biossíntese , Escherichia coli/genética , Metiltransferases/biossíntese , Biodegradação Ambiental , Elastina/genética , Escherichia coli/enzimologia , Engenharia Genética , Metilação , Metiltransferases/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , S-Adenosilmetionina/metabolismo , TemperaturaRESUMO
BACKGROUND: Pseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression. METHODOLOGY: Lens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies. RESULTS: The lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in ß-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation. CONCLUSION: The study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.
Assuntos
Humor Aquoso/metabolismo , Elastina/genética , Síndrome de Exfoliação/genética , Regulação da Expressão Gênica , Homocisteína/genética , Pressão Intraocular/fisiologia , Cristalino/metabolismo , Western Blotting , Elastina/biossíntese , Síndrome de Exfoliação/metabolismo , Homocisteína/biossíntese , Humanos , Estudos Prospectivos , RNA/genéticaRESUMO
INTRODUCTION: An antiaging regimen that aids in clearing the matrix of waste products and stimulating neocollagenesis and neoelastogenesis was tested among a group of subjects over the course of 12 weeks to assess its efficacy in women with mild to moderate wrinkles and skin sagging on the face. MATERIALS AND METHODS: The efficacy of the product regimen was tested in 22 subjects using investigator clinical grading measurements, raking light imaging, 3D imaging, biopsies, and self-assessment questionnaires at baseline and weeks 4, 8, and 12. RESULTS: Clinical grading indicated that use of the antiaging regimen for 12 weeks produced a statistically significant improvement in scores for all evaluated parameters; the raking light image analysis demonstrated a statistically significant improvement in values for length, width, and area of wrinkles when compared with baseline values as did 3D imaging. Biopsy results in the 5 patients tested showed improvement in solar elastosis, collagen stimulation, and improvement in cornified layers in all 5 patients. Elastin stimulation was evident in 3 of 5 patients. Results from the self-assessment questionnaire analysis indicated favorable responses in a statistically significant proportion of subjects after 12 weeks of use for all inquiries. CONCLUSION: Use of this facial antiaging regimen was effective in improving visual facial photoaging conditions and well-perceived when used by women with mild to moderate wrinkles and skin sagging on the face under the conditions of this study.
Assuntos
Peptídeos/uso terapêutico , Envelhecimento da Pele/efeitos dos fármacos , Higiene da Pele/métodos , Pele/diagnóstico por imagem , Idoso , Biópsia , Colágeno/biossíntese , Elastina/biossíntese , Matriz Extracelular/metabolismo , Feminino , Humanos , Imageamento Tridimensional , Luz , Pessoa de Meia-Idade , Satisfação do Paciente , Pele/patologia , Envelhecimento da Pele/patologia , Inquéritos e Questionários , Resultado do TratamentoRESUMO
Silk-elastin-like-protein polymers (SELPs) are genetically engineered recombinant protein sequences consisting of repeating units of silk-like and elastin-like blocks. By combining these entities, it is shown that both the characteristic strength of silk and the temperature-dependent responsiveness of elastin can be leveraged to create an enhanced stimuli-responsive material. It is hypothesized that SELP behavior can be influenced by varying the silk-to-elastin ratio. If the responsiveness of the material at different ratios is significantly different, this would allow for the design of materials with specific temperature-based swelling and mechanical properties. This study demonstrates that SELP fiber properties can be controlled via a temperature transition dependent on the ratio of silk-to-elastin in the material. SELP fibers are experimentally wet spun from polymers with different ratios of silk-to-elastin and conditioned in either a below or above transition temperature (T t ) water bath prior to characterization. The fibers with higher elastin content showed more stimuli-responsive behavior compared to the fibers with lower elastin content in the hot (57-60 °C) versus cold (4-7 °C) environment, both computationally and experimentally. This work builds a foundation for developing SELP materials with well-characterized mechanical properties and responsive features.
Assuntos
Materiais Biocompatíveis/síntese química , Elastina/química , Hidrogéis/química , Proteínas Recombinantes/química , Seda/química , Sequência de Aminoácidos , Animais , Materiais Biocompatíveis/metabolismo , Bombyx/química , Elasticidade , Elastina/biossíntese , Elastina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Ligação de Hidrogênio , Teste de Materiais , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Resistência à TraçãoRESUMO
BACKGROUND: A single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema. METHODS: C57BL/6 mice were randomly divided into control (CTRL) and emphysema (E) groups. Mice in the E group received porcine pancreatic elastase (0.2 IU, 50 µL) intratracheally once weekly for four consecutive weeks; the CTRL animals received sterile saline (50 µL) using the same protocol. Three hours after the last instillation, the E group was further randomized to receive either saline (SAL) or murine MSCs (105 cells) intratracheally, in one or two doses (1 week apart). Fourteen days later, mice were euthanized, and all data analyzed. RESULTS: Both one and two doses of MSCs improved lung mechanics, reducing keratinocyte-derived chemokine and transforming growth factor-ß levels in lung homogenates, total cell and macrophage counts in bronchoalveolar lavage fluid (BALF), and collagen fiber content in airways and blood vessels, as well as increasing vascular endothelial growth factor in lung homogenates and elastic fiber content in lung parenchyma. However, only the two-dose group exhibited reductions in tumor necrosis factor-α in lung tissue, BALF neutrophil and lymphocyte count, thymus weight, and total cellularity, as well as CD8+ cell counts and cervical lymph node CD4+ and CD8+ T cell counts, as well as further increased elastic fiber content in the lung parenchyma and reduced severity of pulmonary arterial hypertension. CONCLUSIONS: Two doses of MSCs enhanced lung repair and improvement in cardiac function, while inducing T cell immunosuppression, mainly of CD8+ cells, in elastase-induced emphysema.
Assuntos
Sistema Cardiovascular/patologia , Pulmão/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Enfisema Pulmonar/terapia , Cicatrização , Animais , Líquido da Lavagem Broncoalveolar , Sistema Cardiovascular/fisiopatologia , Colágeno/metabolismo , Elastina/biossíntese , Feminino , Terapia de Imunossupressão , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/fisiopatologia , Tecido Linfoide/patologia , Camundongos Endogâmicos C57BL , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologiaRESUMO
Elastin and collagen are the two main components of elastic tissues and provide the tissue with elasticity and mechanical strength, respectively. Whereas collagen is adequately produced in vitro, production of elastin in tissue-engineered constructs is often inadequate when engineering elastic tissues. Therefore, elasticity has to be artificially introduced into tissue-engineered scaffolds. The elasticity of scaffold materials can be attributed to either natural sources, when native elastin or recombinant techniques are used to provide natural polymers, or synthetic sources, when polymers are synthesized. While synthetic elastomers often lack the biocompatibility needed for tissue engineering applications, the production of natural materials in adequate amounts or with proper mechanical strength remains a challenge. However, combining natural and synthetic materials to create hybrid components could overcome these issues. This review explains the synthesis, mechanical properties, and structure of native elastin as well as the theories on how this extracellular matrix component provides elasticity in vivo. Furthermore, current methods, ranging from proteins and synthetic polymers to hybrid structures that are being investigated for providing elasticity to tissue engineering constructs, are comprehensively discussed. STATEMENT OF SIGNIFICANCE: Tissue engineered scaffolds are being developed as treatment options for malfunctioning tissues throughout the body. It is essential that the scaffold is a close mimic of the native tissue with regards to both mechanical and biological functionalities. Therefore, the production of elastic scaffolds is of key importance to fabricate tissue engineered scaffolds of the elastic tissues such as heart valves and blood vessels. Combining naturally derived and synthetic materials to reach this goal proves to be an interesting area where a highly tunable material that unites mechanical and biological functionalities can be obtained.
Assuntos
Elasticidade , Polímeros/química , Engenharia Tecidual/métodos , Animais , Elastina/biossíntese , Elastina/química , Humanos , Polímeros/síntese química , Alicerces Teciduais/químicaRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by high levels of protease activity leading to degradation of elastin followed by loss of elasticity of the lung and the development of emphysema. Elastin is an essential structural component of the lung parenchyma to support the expansion and recoil of the alveoli during breathing. The lung extracellular matrix is vulnerable to pathological structural changes upon upregulation of serine proteases, including cathepsin G (CG) and proteinase 3 (PR3). In this study, we explored the diagnostic features of elastin neo-epitopes generated by CG and PR3. Two novel competitive enzyme-linked immunosorbent assays (ELISA) measuring CG and PR3 generated elastin fragments (EL-CG and ELP-3 respectively) were developed for assessment in serum. Both assays were technically robust and biologically validated in serum from patients with COPD. Serological levels of both elastin fragments were significantly elevated in patients with COPD compared to healthy controls. These data suggest that EL-CG and ELP-3 may serve as plausible biologic markers of destructive changes in COPD.