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1.
Am J Respir Cell Mol Biol ; 63(5): 699-706, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32790529

RESUMO

The mechanisms responsible for the increased loss of pulmonary function following acute lung inflammation in chronic obstructive pulmonary disease remain poorly understood. To investigate this process, our laboratory developed a hamster model that uses a single intratracheal instillation of LPS to superimpose an inflammatory response on lungs treated with intratracheal elastase 1 week earlier. Parameters measured at 2 days after LPS included total leukocyte content and percent neutrophils in BAL fluid (BALF), and BALF levels of both total and peptide-free elastin-specific crosslinks, desmosine and isodesmosine (DID). Airspace enlargement, measured by the mean linear intercept method, and relative interstitial elastic fiber surface area were determined at 1 week after LPS. Compared with animals only treated with elastase, those receiving elastase/LPS showed statistically significant increases in mean linear intercept (156.2 vs. 85.5 µm), BALF leukocytes (187 vs. 37.3 × 104 cells), neutrophils (39% vs. 3.4%), and free DID (182% vs. 97% of controls), which exceeded the sum of the individual effects of the two agents. Despite increased elastin breakdown, the elastase/LPS group had significantly greater elastic fiber surface area than controls (49% vs. 26%) owing to fragmentation and splaying of the fibers. Additional experiments showed that the combination of elastin peptides and LPS significantly enhanced their separate effects on BALF neutrophils and BALF DID in vivo and leukocyte chemotaxis in vitro. The results suggest that structural changes in elastic fibers have proinflammatory activity and may contribute to the decline in pulmonary function related to chronic obstructive pulmonary disease exacerbations.


Assuntos
Tecido Elástico/patologia , Inflamação/patologia , Animais , Líquido da Lavagem Broncoalveolar , Quimiotaxia , Desmosina/metabolismo , Elastina/metabolismo , Feminino , Isodesmosina/metabolismo , Leucócitos/citologia , Lipopolissacarídeos , Pulmão/patologia , Masculino , Mesocricetus , Peptídeos/metabolismo
3.
Acta Biomater ; 105: 180-190, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31982591

RESUMO

Intrinsically poor auto-regenerative repair of proteolytically-disrupted elastic matrix structures by resident SMCs in the wall of abdominal aortic aneurysms (AAAs) prevents growth arrest and regression of these wall expansions. Supporting their possible future use in a regenerative cell therapy for AAAs, in a prior study, we showed that bone marrow mesenchymal stem cell-derived Smooth Muscle Cells (BM-SMCs) secrete biological factors that have significant pro-elastogenic and anti-proteolytic effects on aneurysmal rat aortic SMCs (EaRASMCs) in non-contact co-cultures. We also identified one stable BM-SMC phenotype (cBM-SMC) generated by differentiating BM-MSCs on a 2D fibronectin substrate in the presence of PDGF (Platelet Derived Growth Factor) and TGF-ß1 (Transforming Growth Factor-ß1) that exhibited superior elastogenicity and pro-elastogenic/anti-proteolytic properties. In this study, we further investigated the ability of these cBM-SMCs to maintain these superior elastogenic properties in a 3D collagenous milieu alone and in co-culture with EaRASMC to evaluate their potential as an alternative cell source for cell therapy in AAA. Some of our key observations were higher contractility and greater amount of structurally intact elastin production in both standalone culture of cBM-SMCs as well as co-culture of cBM-SMCs with EaRASMCs as shown by VVG (Verhoeff-Van Gieson) staining and Pontamine Sky Blue labeling and lower MMP-9 protein expression in standalone culture in 3D collagenous environment. Our overall result indicates that cBM-SMCs possess the ability to provide elastogenic impetus in a 3D collagenous AAA milieu which is otherwise not conducive to elastogenesis. Therefore our study strongly suggest the utility of cBM-SMCs as a potential cell source for cell therapy to augment elastic matrix neo-assembly and fiber formation and attenuate proteolysis in a collagenous milieu that is evocative of the de-elasticized aneurysmal wall. STATEMENT OF SIGNIFICANCE: Abdominal aortic aneurysm (AAA) or ballooning of the aorta is one of the leading causes of cardiovascular disease (CVD) related death caused by significantly increased proteolytic activity in the aortic wall. Reversing pathophysiology of this condition is challenging due to intrinsically poor regeneration of elastin by aortic smooth muscle cells. Current management of AAA is limited to passive monitoring of the disease until it becomes large enough to receive surgical intervention and no drug based therapy currently exists. Cell based therapy can be a potential alternative treatment in this scenario because it provides elastogenic impetus to the aneurysmal SMCs, compensates for the dead SMCs and serves as a robust source of elastin while being delivered with minimal invasiveness. Hence this work will have significant impact in the field of tissue engineering and regenerative medicine.


Assuntos
Colágeno/farmacologia , Elasticidade , Células-Tronco Mesenquimais/citologia , Miócitos de Músculo Liso/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desmosina/metabolismo , Elastina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fluorescência , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos Sprague-Dawley , Tecidos Suporte/química
4.
Asian J Androl ; 22(5): 485-492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31736474

RESUMO

This study aimed to explore whether and how anti-lysyl oxidase (anti-LOX) combined with a vacuum device (VD) could promote penile lengthening and to evaluate the effect on erectile function. This study was performed on four groups of adult rats: control, anti-LOX, VD (negative pressure value of -300 mmHg), and anti-LOX + VD. Penile length was measured by a modified VD method and verified on exposed length data. Intracavernous pressure (ICP) and maximum ICP/mean arterial pressure (MAP) ratio were recorded to assess erectile function. For corpus cavernosum, LOX activity and concentrations of pyridinoline, desmosine, hydroxyproline, and elastin were analyzed; transmission electron microscope and Hart's elastin staining were performed to monitor microstructural changes. Anti-LOX and VD significantly lengthened the penis by 10.8% (3.75 mm) and 8.2% (2.48 mm) compared with the control group, respectively, while anti-LOX + VD achieved the longest penile size (40.58 ± 0.40 mm) which was 17.4% longer than the control group (34.58 ± 0.54 mm). After 1-week washout, no penile retraction was observed. Meanwhile, exposed penile length data confirmed that the penis in the anti-LOX + VD group was also significantly longer. Anti-LOX inhibited LOX activity to reduce pyridinoline level, which led the penile tunica albuginea remodeling. However, it had no effect on hydroxyproline, desmosine, and elastin levels. Moreover, anti-LOX had no impact on erectile function, which was determined by ICP and ICP/MAP ratio. These results suggest that anti-LOX elongates the penis by reducing pyridinoline, which induces tunica albuginea remodeling. This lengthening effect was more obvious when combined with a VD. All procedures had no impact on erectile function.


Assuntos
Aminopropionitrilo/análogos & derivados , Inibidores Enzimáticos/uso terapêutico , Pênis/anatomia & histologia , Pênis/fisiologia , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Vácuo , Aminoácidos/metabolismo , Aminopropionitrilo/uso terapêutico , Animais , Pressão Arterial , Colágeno/metabolismo , Colágeno/ultraestrutura , Terapia Combinada , Desmosina/metabolismo , Elastina/metabolismo , Elastina/ultraestrutura , Hidroxiprolina/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ereção Peniana , Pênis/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Biomech Model Mechanobiol ; 19(1): 99-112, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31270728

RESUMO

Deposition of elastin and collagen in the aorta correlates with increases in blood pressure and flow during development, suggesting that the aorta adjusts its mechanical properties in response to hemodynamic stresses. Elastin knockout (Eln-/-) mice have high blood pressure and pathological remodeling of the aorta and die soon after birth. We hypothesized that decreasing blood pressure in Eln-/- mice during development may reduce hemodynamic stresses and alleviate pathological remodeling of the aorta. We treated Eln+/+ and Eln-/- mice with the anti-hypertensive medication captopril throughout embryonic development and then evaluated left ventricular (LV) pressure and aortic remodeling at birth. We found that captopril treatment decreased Eln-/- LV pressure to values near Eln+/+ mice and alleviated the wall thickening and changes in mechanical behavior observed in untreated Eln-/- aorta. The changes in thickness and mechanical behavior in captopril-treated Eln-/- aorta were not due to alterations in measured elastin or collagen amounts, but may have been caused by alterations in smooth muscle cell (SMC) properties. We used a constitutive model to understand how changes in stress contributions of each wall component could explain the observed changes in composite mechanical behavior. Our modeling results show that alterations in the collagen natural configuration and SMC properties in the absence of elastin may explain untreated Eln-/- aortic behavior and that partial rescue of the SMC properties may account for captopril-treated Eln-/- aortic behavior.


Assuntos
Aorta/crescimento & desenvolvimento , Captopril/farmacologia , Elastina/deficiência , Estresse Mecânico , Remodelação Vascular/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Aorta/efeitos dos fármacos , Fenômenos Biomecânicos/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Desmosina/metabolismo , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Hidroxiprolina/metabolismo , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo
6.
J Am Heart Assoc ; 8(20): e013743, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31595818

RESUMO

Background It is recognized that factors beyond aortic size are important in predicting outcome in abdominal aortic aneurysm (AAA) disease. AAA is characterized by the breakdown of elastin within the aortic tunica media, leading to aortic dilatation and rupture. The aim of this study was to investigate the association of plasma desmosine (pDES), an elastin-specific degradation product, with disease severity and clinical outcome in patients with AAA. Methods and Results We measured pDES and serum biomarker concentrations in 507 patients with AAAs (94% men; mean age, 72.4±6.1 years; mean AAA diameter, 48±8 mm) and 162 control subjects (100% men; mean age, 71.5±4.4 years) from 2 observational cohort studies. In the longitudinal cohort study (n=239), we explored the incremental prognostic value of pDES on AAA events. pDES was higher in patients with AAA compared with control subjects (mean±SD: 0.46±0.22 versus 0.33±0.16 ng/mL; P<0.001) and had the strongest correlation with AAA diameter (r=0.39; P<0.0001) of any serum biomarker. After adjustment for baseline AAA diameter, pDES was associated with an AAA event (hazard ratio, 2.03 per SD increase [95% CI, 1.02-4.02]; P=0.044). In addition to AAA diameter, pDES provided incremental improvement in risk stratification (continuous net reclassification improvement, 34.4% [95% CI, -10.8% to 57.5%; P=0.09]; integrated discrimination improvement, 0.04 [95% CI, 0.00-0.15; P=0.050]). Conclusions pDES concentrations predict disease severity and clinical outcomes in patients with AAA. Clinical Trial Registration http://www.isrctn.com. Unique identifier: ISRCTN76413758.


Assuntos
Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/sangue , Desmosina/sangue , Idoso , Aneurisma da Aorta Abdominal/diagnóstico , Aneurisma da Aorta Abdominal/epidemiologia , Biomarcadores/sangue , Cateterismo Cardíaco , Feminino , Seguimentos , Humanos , Incidência , Masculino , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida/tendências , Ultrassonografia , Reino Unido/epidemiologia
7.
FEBS J ; 286(18): 3594-3610, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31102572

RESUMO

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner.


Assuntos
Elastina/química , Lisina/química , Peptídeos/química , Tropoelastina/química , Sequência de Aminoácidos/genética , Desmosina/química , Tecido Elástico/química , Tecido Elástico/ultraestrutura , Elastina/ultraestrutura , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Estrutura Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/ultraestrutura , Processamento de Proteína Pós-Traducional/genética , Pele/química , Tropoelastina/ultraestrutura
8.
Arch Biochem Biophys ; 666: 127-137, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30914253

RESUMO

Female reproductive tissues undergo significant alterations during pregnancy, which may compromise the structural integrity of extracellular matrix proteins. Here, we report on modifications of elastic fibers, which are primarily composed of elastin and believed to provide a scaffold to the reproductive tissues, due to parity and parturition. Elastic fibers from the upper vaginal wall of virgin Sprague Dawley rats were investigated and compared to rats having undergone one, three, or more than five pregnancies. Optical microscopy was used to study fiber level changes. Mass spectrometry, 13C and 2H NMR, was applied to study alterations of elastin from the uterine horns. Spectrophotometry was used to measure matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentration changes in the uterine horns. Elastic fibers were found to exhibit increase in tortuosity and fragmentation with increased pregnancies. Surprisingly, secondary structure, dynamics, and crosslinking of elastin from multiparous cohorts appear similar to healthy mammalian tissues, despite fragmentation observed at the fiber level. In contrast, elastic fibers from virgin and single pregnancy cohorts are less fragmented and comprised of elastin exhibiting structure and dynamics distinguishable from multiparous groups, with reduced crosslinking. These alterations were correlated to matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentrations. This work indicates that fiber level alterations resulting from pregnancy and/or parturition, such as fragmentation, rather than secondary structure (e.g. elastin crosslinking density), appear to govern scaffolding characteristics in the female reproductive tissues.


Assuntos
Elastina/química , Paridade/fisiologia , Vagina/metabolismo , Animais , Desmosina/metabolismo , Tecido Elástico/química , Tecido Elástico/metabolismo , Elastina/metabolismo , Feminino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Gravidez , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Inibidor Tecidual de Metaloproteinase-1/metabolismo
9.
Am J Physiol Lung Cell Mol Physiol ; 316(4): L608-L620, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30675803

RESUMO

Deterioration of lung functions and degradation of elastin fibers with age are accelerated during chronic obstructive pulmonary disease (COPD). Excessive genesis of soluble elastin peptides (EP) is a key factor in the pathophysiology of COPD. We have previously demonstrated that 6-wk-old mice exhibited emphysematous structural changes associated with proinflammatory immune response after EP instillation. In this study, we investigated the consequences of aging on inflammatory, immune, and histological criteria associated with murine emphysema progression after EP exposure. Young (6 wk old) and elderly (15 mo old) C57BL/6J mice were endotracheally instilled with EP, and, at various time points after treatment, the inflammatory cell profiles from bronchoalveolar lavage fluids (BALF) and the T-lymphocyte phenotypes, at local and systemic levels, were analyzed by flow cytometry. Lungs were also prepared to allow morphological and histological analysis by confocal microscopy. Elderly mice exhibited an earlier development of pulmonary emphysema, characterized by an increase of the inflammatory and lymphocytic infiltrates, extracellular matrix breakdown, and airspace enlargement compared with young mice. This age-dependent parenchymal tissue remodeling was associated with an increase of the matrix metalloproteinase expressions and desmosine levels in BALF and/or sera of EP-treated mice. In addition, both the proportion of CD4+CD28- and CD8+CD28- T cells in the tissues of EP-treated mice and the interferon-γ levels in the EP-specific memory T-cell clones were significantly higher in elderly versus younger mice. This study demonstrates that aging accelerates emphysema development and that this effect is linked to increased EP production and their effects on inflammatory and immune response.


Assuntos
Envelhecimento/imunologia , Envelhecimento/patologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Desmosina/metabolismo , Modelos Animais de Doenças , Elastina/administração & dosagem , Elastina/metabolismo , Feminino , Inflamação/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/patologia , Proteólise , Enfisema Pulmonar/etiologia
10.
J Biomed Mater Res B Appl Biomater ; 107(5): 1551-1559, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30267643

RESUMO

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H2 O2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H2 O2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Catequina/análogos & derivados , Elastina/química , Elastina/metabolismo , Próteses Valvulares Cardíacas , Valvas Cardíacas/transplante , Benzocaína/química , Benzocaína/metabolismo , Fenômenos Biomecânicos , Bioprótese , Coagulação Sanguínea/efeitos dos fármacos , Catequina/química , Catequina/metabolismo , Cloranfenicol/química , Cloranfenicol/metabolismo , Reagentes para Ligações Cruzadas/química , Desmosina/química , Desmosina/metabolismo , Combinação de Medicamentos , Etildimetilaminopropil Carbodi-Imida/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glutaral/metabolismo , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Nitrofurazona/química , Nitrofurazona/metabolismo , Pericárdio/química
11.
Appl Microbiol Biotechnol ; 102(24): 10485-10494, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30368580

RESUMO

Leather biotechnology based on enzyme is one of the main directions toward clean technology in the leather manufacturing process. Proteins such as collagen, elastin, and keratin are important components in animal hides or skins, and proteases are most frequently used in the leather manufacturing process for the removal of interfibrillar substance and opening-up of collagen fiber instead of toxic chemicals. Elastin is an important and highly elastic structural protein in the animal hides or skins and significantly affects the properties of the final leather product. For improving the quality of leather product, thorough understanding of the mechanism of action of proteases on elastin is necessary. The action of proteases on elastin has been mostly studied either qualitatively by histological analysis or quantitatively based on substrate casein or stained substrates, such as congo red-elastin and Remazol Brilliant Blue R-elastin; however, the resulting products have not been accurately characterized and thus these methods are not up to the standard. Besides, controlling the hydrolytic action of proteases to elastin has been very difficult, and excessive hydrolytic action of protease damages the elastin, restricting the wide application of proteases in the leather manufacturing process. In order to quantitatively evaluate the hydrolytic action of proteases on elastin in a more accurate manner, in this study, a new method was established by determining the unique amino acid desmosine based on the covalently bonded elastin-desmosine conjugate. Quantitative analysis of desmosine was performed in liquor based on cowhides substrate, and qualitative characterization was accomplished by histological analysis of elastic fiber in hides using an optical microscope. The results of this study indicated that the newly developed method is sensitive, accurate, and reproducible. In addition, the unhairing trials also demonstrated the suitability of newly established method in the leather manufacturing process to evaluate the action of proteases on the elastin in animal hides or skins.


Assuntos
Desmosina/análise , Elastina/metabolismo , Peptídeo Hidrolases/metabolismo , Curtume/métodos , Animais , Caseínas/metabolismo , Bovinos , Desmosina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peptídeo Hidrolases/análise , Reprodutibilidade dos Testes , Pele , Temperatura , Fatores de Tempo
12.
Lung ; 196(6): 659-663, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30218154

RESUMO

PURPOSE: While the elastin-specific crosslinks, desmosine and isodesmosine (DID), are increased in blood, urine, and sputum of patients with clinically documented pulmonary emphysema, the usefulness of DID in detecting early lung injury remains untested. To this end, our laboratory has measured DID in a hamster model of smoke-induced emphysema, involving only minimal alveolar wall damage. METHODS: Animals were either treated with cigarette smoke for 2 h/day, 5 days/week, or exposed only to room air (controls) for a period of 3 months. DID levels in bronchoalveolar lavage fluid (BALF) and whole lungs were determined at monthly intervals, using liquid chromatography and tandem mass spectrometry. Lung surface area was also determined, as a measure of airspace enlargement. RESULTS: The portion of BALF DID not bound to peptides (free DID) was significantly higher in smoke-exposed animals at 2 months (9.2 vs 4.4 pg/mg protein; p < 0.05), whereas total BALF DID showed no significant increases over the course of the study, and total lung DID remained unchanged. There was a mild, but significant, loss of lung surface area in the smoke-exposed group at 2 months (28.8% vs 25.2%, p < 0.05), which showed no further progression, consistent with the return of free DID to control levels at 3 months. CONCLUSIONS: These findings support the hypothesis that free DID are sensitive indicators of smoke-induced lung injury. Measurement of free DID in smokers with minimally decreased lung mass may help determine the utility of this parameter as a test for incipient pulmonary emphysema.


Assuntos
Desmosina/metabolismo , Pulmão/metabolismo , Enfisema Pulmonar/metabolismo , Fumar/efeitos adversos , Animais , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Pulmão/patologia , Mesocricetus , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Fatores de Tempo , Regulação para Cima
13.
J Biol Chem ; 293(39): 15107-15119, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30108173

RESUMO

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.


Assuntos
Biopolímeros/química , Elastina/química , Lisina/química , Tropoelastina/química , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/química , Animais , Biopolímeros/genética , Bovinos , Desmosina/química , Dipeptídeos/química , Elastina/genética , Humanos , Domínios Proteicos/genética , Tropoelastina/genética
14.
Anal Bioanal Chem ; 410(26): 6881-6889, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30062515

RESUMO

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.


Assuntos
Desmosina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Desmosina/sangue , Desmosina/química , Desmosina/urina , Humanos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes
15.
Respir Res ; 19(1): 45, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29558926

RESUMO

Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, common initial pathogenic steps have been suggested in both lung diseases. Emphysema is caused by an elastase/anti-elastase imbalance leading to accelerated elastin degradation. Elastinolysis is however, also accelerated in the IPF patients' lungs. The amino acids desmosine and isodesmosine (DES) are unique to elastin. During the degradation process, elastases liberate DES from elastin fibers. Blood DES levels consequently reflect the rate of systemic elastinolysis and are increased in COPD. This is the first report describing elevated DES levels in IPF patients. We also demonstrated that the age-related increment of DES concentrations is enhanced in IPF. Our current study suggests that elastinolysis is a shared pathogenic step in both COPD and IPF. Further investigation is required to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process leads to slower progression of lung fibrosis and better survival for patients with IPF.


Assuntos
Envelhecimento/sangue , Envelhecimento/patologia , Desmosina/sangue , Elastina/metabolismo , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/diagnóstico , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/diagnóstico
16.
Ann Am Thorac Soc ; 15(Suppl 1): S15-S17, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29461896

RESUMO

This article assesses developments in cardiorespiratory medicine since the Nobel Prize in Physiology or Medicine was awarded in 1956 for advancements in the study of cardiorespiratory disease. In chronic obstructive pulmonary disease, advances were accelerated by the discovery of a genetically determined cause for pulmonary emphysema in the genetic abnormality alpha-1 antitrypsin deficiency. This causes a deficiency of the inhibitor of neutrophil elastase, which results in increased degradation of lung elastin and the development of pulmonary emphysema. This discovery gave focus to two amino acids that reside only in body elastin, desmosine and isodesmosine, which can be measured as biomarkers of elastin degradation in body fluids with increased accuracy and sensitivity. Studies of this biomarker have shown that augmentation therapy in alpha-1 antitrypsin deficiency does decrease lung and body elastic tissue degradation and in the RAPID (Randomized, Placebo-controlled Trial of Augmentation Therapy in Alpha-1 Proteinase Inhibitor Deficiency) Study, over 4 years, showed a preservation of lung density by computer tomography correlating with decreases in plasma levels of desmosine and isodesmosine. This insight indicates the potential of agents that prevent lung elastin degradation. Such an agent is hyaluronan aerosol, which is deficient in post mortem lungs with chronic obstructive pulmonary disease and has been shown to block elastin degradation, possibly by a barrier function. Thus it would appear that hyaluronan could have therapeutic potential in chronic obstructive pulmonary disease.


Assuntos
Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/terapia , Enfisema Pulmonar/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Animais , Biomarcadores/metabolismo , Desmosina/metabolismo , Elastina/metabolismo , Humanos , Isodesmosina/metabolismo , Elastase de Leucócito/metabolismo , Enfisema Pulmonar/etiologia , Deficiência de alfa 1-Antitripsina/complicações
17.
Methods Cell Biol ; 143: 133-146, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29310774

RESUMO

Elastin and collagen levels in tissues are frequently difficult to measure because of each protein's limited solubility. This chapter provides detailed methodology for the determination of elastin, collagen, and total protein levels in a single tissue sample. All three assays start with an acid hydrolysate of the tissue, which breaks the tissue-associated proteins down to their component amino acids. Marker amino acids unique to each protein (desmosine for elastin and hydroxyproline for collagen) are then quantified. Total protein content, useful as a denominator for data normalization, can also be measured from a portion of the hydrolysate using an assay for free amino groups. These measurements are performed using convenient 96-well assay plates and require only a plate reader to determine absorbance.


Assuntos
Colágeno/análise , Elastina/análise , Animais , Desmosina/química , Elastina/química , Hidrólise , Hidroxiprolina/química , Solubilidade
18.
Chest ; 153(4): 792-798, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29289686

RESUMO

Insights into the clinical course of COPD indicate the need for new therapies for this condition. The discovery of alpha-1 antitrypsin deficiency (AATD) led to the protease-antiprotease imbalance hypothesis, which was applied to COPD related to AATD as well as COPD not related to AATD. The discovery of AATD brought recognition to the importance of elastin fibers in maintaining lung matrix structure. Two cross-linking amino acids, desmosine and isodesmosine (DI), are unique to mature elastin and can serve as biomarkers of the degradation of elastin. The intravenous augmentation treatment and lung density in severe alpha-1 antitrypsin deficiency (RAPID) study shows a correlation of an anatomic index of COPD (on CT imaging) correlating with a chemical indicator of matrix injury in COPD, DI. The results suggest that preservation of lung elastin structure may slow the progression of COPD. Hyaluronan aerosol decreases the severity of elastase-induced emphysema in animals and has induced reductions in DI levels in preliminary human studies. Hyaluronan deserves further development as a therapy for COPD.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Ácido Hialurônico/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Desmosina/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Imunidade Celular , Isodesmosina/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/imunologia , Ratos
19.
Respirology ; 23(2): 176-181, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28905464

RESUMO

BACKGROUND AND OBJECTIVE: Matrix degradation is a key feature of chronic obstructive pulmonary disease (COPD). Desmosine and isodesmosine (desmosines) are excreted in urine following matrix degradation. The main purpose of this study was to investigate the association between computed tomography (CT) emphysema indices and urinary desmosines in patients with COPD. METHODS: A total of 152 subjects were selected from the Korean Obstructive Lung Disease cohort. Their urine samples were assayed for desmosines using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The cohort was divided into emphysema-dominant (n = 80) and non-emphysema dominant- (n = 72) groups according to the CT emphysema index. RESULTS: The level of urinary desmosines was significantly higher in the emphysema-dominant group. Significant differences were also observed between the two groups for body mass index and lung function. Multivariate analysis indicated that a high level of urinary desmosines was a significant independent predictor of emphysema (relative risk: 2.6; 95% CI: 1.11-6.09; P = 0.028). The percentage of frequent exacerbators was significantly higher in the high urinary desmosine group in the first year of follow-up (P = 0.041). The mean number of exacerbations was higher in the high urinary desmosine group, although this difference was not statistically significant (P = 0.067). The changes in emphysema index did not differ between the two urinary desmosine groups over 3 years of follow-up. CONCLUSION: This study indicates that the level of urinary desmosines measured by LC-MS/MS methods is associated with the CT emphysema index. Urinary desmosine can be a useful predictor in identifying frequent exacerbators.


Assuntos
Desmosina/urina , Isodesmosina/urina , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/urina , Enfisema Pulmonar/urina , Idoso , Biomarcadores/urina , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/etiologia , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios X
20.
Med Hypotheses ; 108: 38-41, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29055397

RESUMO

Elastin is a unique protein providing deformability and resilience to dynamic tissues, such as arteries and lungs. It is an absolute basic requirement for circulation and respiration. Elastin can be degraded by elastases and has a high calcium affinity. Elastin calcification and elastin degradation are two pathological processes that impair elastin's functioning. Furthermore, elastin degradation can be associated to elastin calcification. Matrix Gla Protein (MGP) is probably the most potent natural inhibitor of elastin calcification and requires vitamin K for its activation. Measuring circulating levels of inactive MGP (dp-ucMGP) is a frequently used method to assess vitamin K status. Dp-ucMGP reflects the burden of vitamin K-dependent proteins that have not been activated by vitamin K and could therefore best be regarded as a biomarker of a vitamin K deficit. Dp-ucMGP levels decrease after vitamin K supplementation. Since the amino acids desmosine and isodesmosine (DES) are unique to crosslinked elastin fibers, systemic elastin degradation can be assessed with the plasma DES assay. Recently, we discovered a strong correlation between plasma dp-ucMGP and plasma DES levels in both patients with chronic obstructive pulmonary disease (COPD) and controls. The 'Vitamin K deficit and elastolysis theory' posits that elastin degradation causes a rise in the vitamin K deficit and implies that vitamin K supplementation could be preventing elastin degradation. If this hypothesis holds true and is universally found in every state and condition, it will have an unprecedented impact on the management of every single pulmonary disease characterized by accelerated elastin degradation, such as alpha-1 antitrypsin deficiency, bronchiectasis, COPD and cystic fibrosis. Theoretically, a plasma dp-ucMGP concentration of zero would be associated with a near-complete standstill of elastin degradation and disease progression in patients with any of these debilitating conditions.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Pneumopatias/metabolismo , Deficiência de Vitamina K/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Desmosina/sangue , Elasticidade , Humanos , Isodesmosina/sangue , Modelos Biológicos , Vitamina K/uso terapêutico , alfa 1-Antitripsina/metabolismo
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