Elastin is an essential extracellular protein that is a key component of elastic fibres, providing elasticity to cardiac, dermal, and arterial tissues. During the development of the human cardiovascular system, elastin self-assembles before being integrated into fibres, undergoing no significant turnover during the human lifetime. Abnormalities in elastin can adversely affect its self-assembly, and may lead to malformed elastic fibres. Due to the longevity required of these fibres, even minor abnormalities may have a large cumulative effect over the course of a lifetime, leading to late-onset vascular diseases. This thesis project has identified important, over-represented repetitive elements in elastin which are believed to be important for the self-assembly and elastomeric properties of elastin. Initial studies of single nucleotide polymorphisms (SNPs) from the HapMap project and dbSNP resulted in a set of genetic variation sites in the elastin gene. Based on these studies, glycine to serine and lysine to arginine substitutions were introduced in elastin-like polypeptides. The self-assembly properties of the resulting elastin-like polypeptides were observed under microscope and measured using absorbance at 440nm. Assembled polypeptides were also cross-linked to form thin membranes whose mechanical and physical properties were measured and compared. These mutations resulted in markedly different behavior than wild-type elastin-like proteins, suggesting that mutations in the repetitive elements of the elastin sequence can lead to adverse changes in the physical and functional properties of the resulting protein. Using next-generation sequencing, patients with thoracic aortic aneurysms are being genotyped to discover polymorphisms which may adversely affect the self-assembly properties of elastin, providing a link between genetic variation in elastin and cardiovascular disease.

Aachen, Techn. Hochsch., Thesis (doctoral), 2002.

The extracellular matrix (ECM) is the material found surrounding the cells in a tissue. One component of the ECM is the elastic fiber, which confers the property of elasticity to its environment. Organs such as the lung, skin and major blood vessels have an abundance of elastic fibers so that they are able to expand and recoil. Elastic fibers are composed of two main components; elastin and microfibrils. Microfibrils are composed primarily of fibrillin-1 and provide a scaffold unto which tropoelastin monomers assemble. Elastic fibers interact with many other proteins in the ECM, one of which is fibulin-5. Based on the severe elastic fiber defects observed in the fibulin-5 null mouse, it was established that fibulin-5 plays an essential role in elastic fiber development. This role may be in the deposition of tropoelastin onto microfibrils and/or in stabilizing the elastic fibers in the extracellular matrix. In the present study, the relationship between fibulin-5 and the elastic fiber was investigated through a number of in vivo and in vitro experiments. To test the hypothesis that fibulin-5 requires the presence of elastin to assemble in the ECM, full-length recombinant fibulin-5 (rF5) was purified from transfected cells and used to make a fibulin-5 antibody. Solid-phase binding assays using rF5 showed that fibulin-5 binds tropoelastin at two sites; the initial portion of the C-terminus and the first calcium-binding epidermal growth factor-like domain at the N-terminus. Immunofluorescence staining of elastin null mouse embryonic fibroblast cultures revealed that fibulin-5 does not require elastin to be present in the ECM in order to assemble. Subsequently, solid-phase binding assays showed that fibulin-5 can bind to the N-terminus of fibrillin-1. To determine if fibulin-5 could exist independent of elastin and/or fibrillin-1 in vivo, an immunohistochemical analysis was conducted on heart, liver, lung, colon, spleen, testis and kidney. All three proteins were co-localized in all organs except in the kidney, where fibrillin-1 was found to independently stain the capillary tufts of the renal corpuscles and renal tubules. Thus, fibulin-5 may be co-regulated with elastin and is not present on elastin-independent microfibrils. Additionally, novel locations of elastic fibers were uncovered in the heart, liver, colon, spleen and testis. Overall, this study provides important insights as to the role of fibulin-5 in elastic fiber structure and assembly and also reveals the complexity in understanding the pathogenesis of diseases involving elastic fiber proteins.

Thesis (Ph. D.)--University of Adelaide, Dept. of Pathology, 1985. / Includes bibliographical references (leaves 266-303).

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13266号 / 論医博第2180号 / 新制||医||1038(附属図書館) / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Despite the availability of a yearly vaccine and antivirals, the incidence of influenza infections remains high. The genome of the influenza virus can mutate rapidly, therefore novel influenza strains that may be resistant to the current vaccine or antivirals frequently enter the population. Because of the long production time necessary to produce a vaccine, new antivirals must be created to combat early stages of influenza outbreaks. The most effective antivirals will target a highly conserved and essential stage of virus replication. The influenza RNA-dependent RNA polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. The three components of the polymerase interact through well-defined domains and are essential for viral replication. Previously, influenza replication has been inhibited using a small synthetic peptide that mimics the interaction domain between PA and PB1 and inhibits the formation of the heterotrimeric complex. Problem and Hypothesis: Although the peptide could inhibit influenza replication, synthetic peptides are costly to produce and are not a viable option for large-scale production. This problem can often be overcome by attaching the peptide to a highly soluble carrier protein. We hypothesize that influenza replication can be inhibited by attaching a peptide, that mimics the binding domain between the PA and PB1 subunits, to a human elastin-like polypeptide (ELP) carrier protein. Methods and Results: The peptide and a nuclear localization sequence was genetically linked to a maltose binding protein (MBP) or ELP carrier protein. The MBP construct was purified by affinity chromatography using FPLC. A high yield of the ELP construct was obtained using inverse transition cycling, a method unique to ELPs because of their temperature-dependent solubility. The ELP construct was designed to be soluble at physiological temperature to limit cellular toxicity due to protein aggregation. The cytotoxicity of the ELP construct was assessed by monitoring the growth of A549 cells, a human lung epithelial cell line. The ELP construct did not have any adverse effects on A549 cell growth. Both constructs could localize to cell nuclei using their respective nuclear localization sequences and could also interact with the PA subunit, demonstrating their potential to inhibit influenza replication. Despite this, only the MBP construct was able to inhibit the replication of influenza. The MBP construct could inhibit the replication of both the H1N1 and H3N2 subtypes of influenza, indicating the recombinant protein had cross-strain activity. Conclusion: Linking a small peptide to carrier protein can result in high protein yields, however a carrier protein must be chosen that will maintain the peptides’ therapeutic activity. In this study, a small anti-influenza peptide inhibited influenza replication when attached to an MBP carrier protein, however was not able to inhibit influenza replication when attached to an ELP carrier protein. Although the peptide was ineffective when attached to this particular ELP carrier protein, different ELP proteins of various lengths and compositions may still be effective carrier proteins for an antiviral peptide. / Thesis / Master of Science (MSc) / The influenza virus causes seasonal outbreaks each year and can have life-threatening symptoms in the young and elderly. In addition, it can rapidly mutate through antigenic drift; therefore, a new vaccine is required each year. Pandemic influenza strains can enter the population when the virus undergoes genetic reassortment by antigenic shift. However, it can take a significant amount of time to formulate a vaccine against pandemic influenzas, which means antiviral drugs are often used as the first line of defense. New antivirals to treat influenza must be developed because resistance to the current influenza antivirals has steadily increased. In this work, we developed an antiviral peptide to disrupt a critical interaction in the influenza RNA-dependent RNA polymerase and inhibit virus replication. This peptide was previously conjugated to an E. coli MBP carrier protein, which would likely not be compatible in vivo. This thesis focused on attaching the antiviral peptide to an elastin-like polypeptide protein, which mimics human tropoelastin, and should be non-immunogenic in humans.

Small diameter synthetic vascular grafts have yet to be clinically successful due to luminal narrowing from thrombosis and intimal hyperplasia. Current attempts to address this issue include the development of materials that support endothelialisation and protein modification to the material surfaces that reduce thrombosis. The extracellular matrix protein elastin has been found to be one of the least thrombogenic components of blood vessels, and its purified and recombinant forms have shown reduced thrombogenicity in both in vitro and in vivo models. Biomaterial coatings of elastin-like polypeptides (ELPs) recombinantly produced in the Woodhouse laboratory showed reduced fibrinogen adsorption, platelet adhesion, and platelet activity. However, the reason for their relative non-thrombogenicity is still not fully understood. In this work, the leukocyte response to ELP-coated materials was investigated. In particular, ELP1 and ELP4, which differ in molecular weight and sequence length, were physically adsorbed to a polyethylene terephthalate surface (MylarTM), yielding 0.22 ± 0.13 μg/cm2 and 0.37 ± 0.19 μg/cm2 surface coverage, respectively, as determined by the colorimetric assay, FastinTM Elastin. These surfaces were exposed to flowing citrated whole blood for surface and bulk evaluation of leukocyte activity using scanning electron microscopy and flow cytometry, respectively. Little leukocyte activation was observed on the surface of the controls, low-density polyethylene and uncoated MylarTM. In the bulk, tissue factor (TF) expression (monocytes: ELP1 = 38.6 ± 16.3 %, ELP4 = 33.9 ± 18.1 %) and platelet-leukocyte aggregates determined by CD61 (monocytes: ELP1 = 63.1 ± 17.1 %, ELP4 = 61.8 ± 16.8 %; granulocytes: ELP1 = 62.7 ± 17.0 %, ELP4 = 60.5 ± 20.1 %) were both decreased compared to uncoated MylarTM, while CD11b upregulation (monocytes: ELP1 = 18.7 ± 2.2 %, ELP4 = 19.7 ± 2.7 %; granulocytes: ELP = 21.4 ± 3.7 %, ELP4 = 22.0 ± 3.2 %) was increased. The statistical dependence of TF expression and platelet-monocyte aggregates was tested; however, no correlation was found. Overall, platelet-leukocyte aggregate formation was reduced and there were conflicting results with regards to the reduction of leukocyte activation for the ELP coatings on MylarTM. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-10-10 15:34:51.802

The development of small-diameter artificial blood vessels that mimic the properties of natural blood vessels has proven to be a clinical challenge. While autologous vessels are the standard, they can be difficult to obtain and require invasive surgeries. Synthetic materials have been successful in large diameter applications, but they have been unsuccessful in small-caliber environments due to a number of factors including thrombus formation, intimal hyperplasia, and infection. Intimal hyperplasia, of particular interest in this study, involves the build up of smooth muscle cells (SMCs) in the intimal layer of the artery due to abnormal migration and proliferation. This work focuses on the development of a new polymer that has the potential to function as an intimal/medial component of a small-diameter blood vessel. Using recombinant elastin-like polypeptides (ELPs) developed by the Woodhouse laboratory, as well as chondroitin sulphate-specific binding sequences (CSBD1 and CSBD2) determined by the Panitch laboratory, a new elastin-like polypeptide-chondroitin sulphate binding domain (ELP-CSBD) block copolymer has been developed and characterized. The expression of the ELP1-CSBDs was accomplished using E. coli BL21 cells in a bioreactor or shaker flask systems. The polypeptides were purified using dialysis and ion exchange chromatography and expression and purity were characterized using mass spectrometry and amino acid analysis. Both ELP1-CSBDs were successfully expressed using these methods and ELP1-CSBD1 was produced to high purities. ELP1-CSBD1 was able to undergo coacervation in vitro, suggesting that ELP1-CSBD1 is able to self-assemble in a manner similar to native elastin. In the presence of the glycosaminoglycan chondroitin sulphate (CS), the temperature of coacervation of ELP1-CSBD1 is increased, the rate and extent of coacervation is decreased, and aggregates remain in solution even at higher temperatures. The influence of heparin was also explored as previous studies indicated that the CS binding domains were shown to also bind to heparin. Studies completed in the presence of heparin showed that there were no significant changes to the coacervation characteristics of ELP1-CSBD1. It is anticipated that when combined with CS, ELP1-CSBD1 will gel, forming a basis for an intimal/medial layer of a TEBV that will modulate SMC response and increase graft integrity. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-01-06 21:03:37.788

Background: There is growing evidence that genetic variants have an impact on the pathogenesis of intracranial aneurysm (IA). Recently, the genetic locus around the elastin gene (7q11) has been identified as linked to IA in a Japanese population. Our aim was to confirm these results in Caucasian populations. Methods: We conducted a case-control study in 120 Caucasian patients with IA and 172 controls to investigate 8 single nucleotide polymorphisms (SNPs) and various haplotypes within the elastin gene, which were frequently found and associated with the phenotype in the Japanese populations. Real-time PCR and melting curve analysis were used for the detection of genotypes. Results: Allele frequencies and genotypes were equally distributed between Caucasian cases and controls. We failed to identify haplotypes that are associated with the phenotype in our population, which is in contrast to the Japanese study. However, allele frequencies in control populations differ between Caucasians and Japanese. Conclusions: We found no association between SNPs and haplotypes of the elastin gene and the occurrence of IA in our Caucasian populations. However, our data provide strong evidence for racial/ethnic differences in the association of SNP and specific haplotypes of the elastin gene with the phenotype. There might be other genetic variants of the elastin gene associated with IA in Caucasians. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

This thesis investigates the mechanical properties of the elastic proteins isolated by cyanogen bromide digestion from lamprey cartilages and compares them with the mammalian protein, elastin. Thermomechanical testing and measurements of the effects of hydrophobic solvents on mechanics are used to determine the energetic and entropic contributions to the mechanical properties and the role of solvent interactions. Raman microspectrometry is shown to be a valuable tool in determining the secondary structure of the proteins, their interactions with water and molecular-level effects of mechanical strain. The supramolecular structure of the proteins matrices are investigated using nonlinear microscopy and X-ray diffraction. The mechanical properties of fibrous elastin agreed with those previously reported with elastic moduli in the region of 0.2-0.4 MPa. Elastic moduli decrease by approximately 25% with increased temperature, which was accompanied by a small decrease in hysteresis loss. In agreement with earlier findings, an entropic mechanism of elasticity became dominant only at high temperatures with a major contribution from interactions with solvent water. The lamprey proteins can be divided into two broad groups, the 'soft' branchial and pericardial cartilages resembling elastin, with linear stress-strain behaviour over a range of strains, elastic moduli in the range 0.13 MPa to 0.35 MPa, breaking strains of up to 50% and low hysteresis. Annular and piston proteins showed a very different response having much higher elastic moduli (0.27 MPa to 0.75 MPa), higher breaking strains and large hysteresis. Similarities between elastin and the lamprey matrix proteins extended to their thermomechanical behaviour with a decrease in elastic moduli and a drive towards entropic elasticity at high temperatures, although the annulus and piston were less thermally stable. Raman spectroscopy was able to detect differences between the various proteins and between elastin fibres and fragmentation products. Although no vibrational modes associated with cross-linking of the fibres could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from \alpha -elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% \beta -structures, 46% unordered and 18% \alpha -helix. \alpha -Elastin contained 29% \beta -structures, 53% unordered and 18% \alpha -helix. Strains of up to 60% in ligament fibre bundles resulted in no significant shifts in peak positions or in secondary structure. Polarization measurements revealed that the peptide bonds and several of the bulky side-chains re-orientated closer to the fibre axis with strain. Heating nuchal elastin fibres to 60^{\circ} C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of \beta -structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water. The Raman spectra of the lamprey matrix proteins are similar both to each other and when compared to fibrous elastin. Only small differences could be detected in side-chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were also very similar to that of elastin, with a preponderance of unordered structures which probably confer the high degree of conformational flexibility necessary for entropy elasticity. Piston and annular proteins, like elastin, showed a strong interaction with water, suggesting a greater role of hydrophobic interactions in their mechanics compared to the branchial and pericardial proteins. Elastin is well known to exhibit autofluorescence. However, only the branchial protein has been reported to autofluoresce. This study shows that all four lamprey matrix proteins investigated exhibit strong autofluorescence which was subsequently exploited to image these tissues using multiphoton microscopy. Microscopic investigations revealed that the architecture of lamprey proteins differ from that of elastin. Nuchal elastin forms bundles of fibres running predominantly parallel to the direction of applied force. The arrangement in lamprey cartilage is very different forming honeycomb structures, which in the case of annular and piston cartilages, is surrounded by a dense sheath of matrix material. Dye injections revealed that the branchial and pericardial form open systems whereas in piston and annular cartilages a closed system exists. These variations in architecture are reflected in their different mechanical properties and in vivo functions.