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Journal of Cardiovascular Pharmacology Apr 2016This study was designed to determine whether vonapanitase (formerly PRT-201), a recombinant human elastase, treatment can fragment the protein elastin in elastic fibers...
PURPOSE
This study was designed to determine whether vonapanitase (formerly PRT-201), a recombinant human elastase, treatment can fragment the protein elastin in elastic fibers and cause dilation of atherosclerotic human peripheral arteries subjected to ex vivo balloon angioplasty.
MATERIALS AND METHODS
Seven patients undergoing lower limb amputation for peripheral artery disease or who died and donated their bodies to science donated 11 tibial arteries (5 anterior, 6 posterior) for this study. All arteries were atherosclerotic by visual inspection. The arteries underwent ex vivo balloon angioplasty and thereafter were cut into rings and studied on wire myographs where the rings were stretched and tension was recorded. After treatment with vonapanitase 2 mg/mL or vehicle control, myography was repeated and the rings were then subject to elastin content measurement using a desmosine radioimmunoassay and elastic fiber visualization by histology. The wire myography data were used to derive compliance, stress-strain, and incremental elastic modulus curves.
RESULTS
Vonapanitase treatment reduced elastin (desmosine) content by 60% and decreased elastic fiber histologic staining. Vonapanitase-treated rings experienced less tension at any level of stretch and as a result had shifts in the compliance and stress-strain curves relative to vehicle-treated rings. Vonapanitase treatment did not alter the incremental elastic modulus curve.
CONCLUSIONS
Vonapanitase treatment of atherosclerotic human peripheral arteries after ex vivo balloon angioplasty fragmented elastin in elastic fibers, decreased tension in the rings at any level of stretch, and altered the compliance and stress-strain curves in a manner predicting arterial dilation in vivo. Based on this result, local treatment of balloon angioplasty sites may increase blood vessel diameter and thereby improve the success of balloon angioplasty in peripheral artery disease.
Topics: Aged; Aged, 80 and over; Angioplasty, Balloon; Atherosclerosis; Carrier Proteins; Elastic Modulus; Elastic Tissue; Elastin; Female; Humans; Male; Middle Aged; Myography; Pancreatic Elastase; Peripheral Arterial Disease; Tibial Arteries; Vasodilation
PubMed: 26745001
DOI: 10.1097/FJC.0000000000000354 -
The Journal of Biological Chemistry Jan 2016MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the...
MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.
Topics: Amino Acid Substitution; Animals; Carrier Proteins; Desmosine; Elastic Tissue; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Glycoproteins; Humans; Ligands; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Microfibrils; Microfilament Proteins; Models, Molecular; Mutation; Peptide Fragments; Protein Interaction Domains and Motifs; Protein Isoforms; Protein Multimerization; Protein Transport; Recombinant Proteins; Tropoelastin
PubMed: 26601954
DOI: 10.1074/jbc.M115.681775 -
American Journal of Physiology. Lung... Jan 2016Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of...
Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII β-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.
Topics: Animals; Bronchoalveolar Lavage Fluid; Collagen; Disease Models, Animal; Elastin; Mice, Inbred C57BL; Pancreatic Elastase; Peptides; Pulmonary Emphysema; Receptors, Cell Surface
PubMed: 26519205
DOI: 10.1152/ajplung.00068.2015 -
Human Molecular Genetics Nov 2015In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can...
In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.
Topics: Amino Acid Oxidoreductases; Animals; Aorta; Cleft Palate; Collagen; Craniofacial Abnormalities; Desmosine; Diaphragm; Embryonic Development; Eye; Gene Targeting; Hydroxyproline; Lung; Mice; Mice, Inbred C57BL; Myocardium; Palate; Spine; Thoracic Vertebrae; Trachea
PubMed: 26307084
DOI: 10.1093/hmg/ddv333 -
The Journal of Biological Chemistry Aug 2015Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause...
Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Blood Vessels; Bone and Bones; Collagen Type I; Cross-Linking Reagents; Cutis Laxa; Disease Models, Animal; Elastic Tissue; Extracellular Matrix Proteins; Fibroblasts; Forelimb; Gene Knock-In Techniques; HEK293 Cells; Humans; Mice, Inbred C57BL; Models, Biological; Molecular Sequence Data; Mutation; Protein Biosynthesis; Protein Multimerization; Protein-Lysine 6-Oxidase; Radiography; Skin; Tendons
PubMed: 26178373
DOI: 10.1074/jbc.M115.640425 -
Biophysical Journal Apr 2015Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses...
Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid-state NMR spectroscopic and relaxation methodologies. Results from macroscopic stress-strain tests are also presented and indicate that glucose-treated elastin is mechanically stiffer than the same tissue without glucose treatment. These measurements show a large hysteresis in the stress-strain behavior of glucose-treated elastin-a well-known signature of viscoelasticity. Two-dimensional relaxation NMR methods were used to investigate the correlation time, distribution, and population of water in these samples. Differences are observed between the relative populations of water, whereas the measured correlation times of tumbling motion of water across the samples were similar. (13)C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after glucose treatment. Although some overall structure is preserved, the process of glucose exposure results in more heterogeneous structures and slower mobility. The correlation times of tumbling motion of the (13)C-(1)H internuclear vectors in the glucose-treated sample are larger than in untreated samples, pointing to their more rigid structure. The (13)C cross-polarization spectra reveal a notably increased α-helical character in the alanine motifs after glucose exposure. Results from molecular dynamics simulations are provided that add further insight into dynamical and structural changes of a short repeat, [VPGVG]5, an alanine pentamer, desmosine, and isodesmosine sites with and without glucose. The simulations point to changes in the entropic and energetic contributions in the retractive forces of VPGVG and AAAAA motifs. The most notable change is the increase of the energetic contribution in the retractive force due to peptide-glucose interactions of the VPGVG motif, which may play an important role in the observed stiffening in glucose-treated elastin.
Topics: Amino Acid Motifs; Animals; Aorta; Elasticity; Elastin; Glucose; Molecular Dynamics Simulation; Protein Binding; Protein Structure, Tertiary; Swine; Viscosity
PubMed: 25863067
DOI: 10.1016/j.bpj.2015.02.005 -
Microbiology (Reading, England) Jul 2015Previous studies have demonstrated that Pseudomonas aeruginosa PAO1 is chemotactic towards proteinogenic amino acids, however, the chemotaxis response of this strain...
Previous studies have demonstrated that Pseudomonas aeruginosa PAO1 is chemotactic towards proteinogenic amino acids, however, the chemotaxis response of this strain towards non-proteinogenic amino acids and the specific chemoreceptors involved in this response are essentially unknown. In this study, we analysed the chemotactic response of PAO1 towards two degradation products of elastin, the lysine-rich, non-proteinogenic amino acids, desmosine and isodesmosine. We observed that isodesmosine, a potential biomarker for different diseases, served as a chemoattractant for PAO1. A screen of 251methyl-accepting chemotaxis proteins mutants of PAO1 identified PctA as the chemoreceptor for isodesmosine. We also showed that the positive chemotactic response to isodesmosine is potentially common by demonstrating chemoattraction in 12 of 15 diverse (in terms of source of isolation) clinical isolates, suggesting that the chemotactic response to this non-proteinogenic amino acid might be a conserved feature of acute infection isolates and thus could influence the colonization of potential infection sites.
Topics: Bacterial Proteins; Chemotactic Factors; Chemotaxis; Desmosine; Elastin; Humans; Isodesmosine; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 25855762
DOI: 10.1099/mic.0.000090 -
American Journal of Physiology. Lung... Jun 2015Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking...
Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia.
Topics: Aminopropionitrile; Animals; Bronchopulmonary Dysplasia; Collagen; Elastin; Extracellular Matrix; Hyperoxia; Lung; Mice; Protein Processing, Post-Translational; Protein-Lysine 6-Oxidase
PubMed: 25840994
DOI: 10.1152/ajplung.00039.2015 -
PloS One 2015The adaptor protein p66Shc regulates intracellular oxidant levels through the modulation of a forkhead-related transcription factor (FOXO3a). The genetic ablation of...
The adaptor protein p66Shc regulates intracellular oxidant levels through the modulation of a forkhead-related transcription factor (FOXO3a). The genetic ablation of p66Shc (p66Shc-/-) renders mice resistant to oxidative stress and p53-dependent apoptosis. We investigated whether p66Shc ablation in mice modifies lung cellular and molecular responses to cigarette smoke (CS) exposure. No differences between wild type (WT) and p66Shc-/- mice were observed in terms of inflammation and oxidant burden after acute CS exposure; however,p66Shc ablation modifies specific features of chronic inflammation induced by repeated exposure to CS. Unlike WT mice, p66Shc-/- mice did not develop emphysema, showing protection toward oxidative damage to DNA and apoptosis as revealed by a trivial 8-hydroxyguanosine staining and faint TUNEL and caspase-3 positivity on alveolar epithelial cells. Unexpectedly, CS exposure in p66Shc-/- mice resulted in respiratory bronchiolitis with fibrosis in surrounded alveoli. Respiratory bronchiolitis was characterized by peribronchiolar infiltrates of lymphocytes and histiocytes, accumulation of ageing pigmented macrophages within and around bronchioles, and peribronchiolar fibrosis. The blockage of apoptosis interferes with the macrophage "clearance" from alveolar spaces, favouring the accumulation of aging macrophages into alveoli and the progressive accumulation of iron pigment in long-lived senescent cells. The presence of areas of interstitial and alveolar fibrosis in peripheral parenchyma often accompanied the bronchiolar changes. Macrophages from smoking p66Shc-/- mice elaborate M2 cytokines (i.e., IL-4 and IL-13) and enzymes (i.e., chitinase and arginase I), which can promote TGF-beta expression, collagen deposition, and fibrosis in the surrounding areas. We demonstrate here that resistance to oxidative stress and p53-dependent apoptosis can modify tissue responses to CS caused by chronic inflammation without influencing early inflammatory response to CS exposure.
Topics: Animals; Apoptosis; Arginase; Bronchiolitis; Chitinases; Desmosine; Fibrosis; Hydroxyproline; Interleukin-13; Interleukin-4; Lung; Macrophages; Mice; Mice, Knockout; Oxidative Stress; Oxidoreductases; Pulmonary Emphysema; Shc Signaling Adaptor Proteins; Smoking; Src Homology 2 Domain-Containing, Transforming Protein 1; Transforming Growth Factor beta; Tumor Suppressor Protein p53
PubMed: 25790295
DOI: 10.1371/journal.pone.0119797 -
Biochimica Et Biophysica Acta May 2015Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH... (Comparative Study)
Comparative Study
Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported.
Topics: Amino Acid Sequence; Animals; Carbon-13 Magnetic Resonance Spectroscopy; Cattle; Elastin; Kinetics; Protein Structure, Tertiary; Sequence Analysis, Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature
PubMed: 25592991
DOI: 10.1016/j.bbapap.2014.12.024