Thesis (Ph. D.)--Columbia University, 1938. / Vita. "Reprinted from the Journal of biological chemistry, 125 ... (1938)." Bibliography: p. 16.

The swelling-temperature compensation hypothesis as proposed by Gosline and French (1979) is examined by investigating the physical and mechanical properties of an evolutionary series of vertebrate elastins. Temperature-dependent swelling, low water contents and thermodynamics typical of hydrophobic systems were observed for all elastins except salmon. Salmon elastin, on the other hand, showed temperature-independent swelling and a high water content. Thermodynamic analysis showed that salmon elastin still contained a hydrophobic component. The swelling-temperature compensation hypothesis suggests that the extreme hydrophobic nature of elastin evolved in order to provide the proteins with temperature-dependent swelling and thus, maintaining elastic efficiency over a wide temperature range. All elastins should then be very hydrophobic systems which is inconsistent with the physical chemical results. All vertebrate elastins are not necessarily hydrophobic systems since salmon elastin shows no temperature-dependence to its swelling. The efficiency of a series of vertebrate elastins was measured over 0 to 60°C temperature range using a forced vibration technique. Over a wide frequency range, both lower and higher vertebrate elastins were capable of efficient spring-like behaviour. Both a higher vertebrate, with temperature-dependent swelling, and a lower vertebrate, with no temperaturate-dependence to its swelling, showed elastic efficiency. The swelling-temperature compensation hypothesis must be rejected. / Science, Faculty of / Zoology, Department of / Graduate

Elastic fibers are major components of ECM, providing tissues with resilience and elastic recoil. They consist of the insoluble elastin made of cross-linked precursor tropoelastin monomers and the microfibrillar scaffold. Various connective tissue disorders have been linked to elastic fiber malformation. In addition, it has been shown that decreased vascular elastin contents correlate with the rapid progression of atherosclerosis in patients with diabetes mellitus. However, it is unknown whether insulin can directly modulate elastogenesis. This question was addressed in this thesis. Our study revealed that insulin stimulated elastogenesis in cultures of human aortic SMCs and skin fibroblasts, and that this effect of insulin was transduced through the insulin receptor. We found that insulin might initiate elastin gene expression and enhance tropoelastin secretion. We also presented novel preliminary data indicating that Glut10 might bind to a fraction of intracellular tropoelastin, and that insulin might modulate the association between these two proteins.

Elastic fibers are major components of ECM, providing tissues with resilience and elastic recoil. They consist of the insoluble elastin made of cross-linked precursor tropoelastin monomers and the microfibrillar scaffold. Various connective tissue disorders have been linked to elastic fiber malformation. In addition, it has been shown that decreased vascular elastin contents correlate with the rapid progression of atherosclerosis in patients with diabetes mellitus. However, it is unknown whether insulin can directly modulate elastogenesis. This question was addressed in this thesis. Our study revealed that insulin stimulated elastogenesis in cultures of human aortic SMCs and skin fibroblasts, and that this effect of insulin was transduced through the insulin receptor. We found that insulin might initiate elastin gene expression and enhance tropoelastin secretion. We also presented novel preliminary data indicating that Glut10 might bind to a fraction of intracellular tropoelastin, and that insulin might modulate the association between these two proteins.

The purpose of this work was to examine the behaviour of the soluble elastin derivative, alpha-elastin, under a variety of conditions.

acase@tulane.edu / Progress towards understanding the underlying mechanisms of pelvic organ prolapse (POP) is limited, in part, due to a lack of information on the biomechanical properties and microstructural composition of the vaginal wall. Compromised vaginal wall integrity is thought to contribute to pelvic floor disorders. In particular, disruption of the elastin metabolism within the vaginal wall extracellular matrix has been highly implicated in POP pathogenesis; however, the role of elastin within the vaginal wall is not fully understood. In addition to the information produced from uniaxial testing, biaxial extension-inflation tests performed over a range of physiological values could provide additional insights into vaginal wall mechanical behavior (i.e. axial coupling and anisotropy) while preserving in vivo tissue geometry. Thus, the objective of this study is to identify the role of elastin in vaginal wall mechanics using physiologically relevant experimental and mathematical approaches. Our specific aims are thus: 1. Develop biaxial mechanical testing methods for assessing the mechanical properties of the murine vaginal wall in a physiological manner. 2. Establish a microstructurally-motivated constitutive model capable of describing the biaxial extension-inflation response of the nonpregnant murine vaginal wall. 3. Quantify the role of elastin in murine vaginal mechanical properties through enzymatic digestion of elastin with elastase.Vaginal tissue from female C57BL/6 mice underwent pressure-diameter and force-length preconditioning and testing within a pressure myograph device before and after elastase digestion. In order to mathematically interpret biaxial data, vaginal tissue was modeled using a 2D membrane approach. Several constitutive models were evaluated on their ability to describe vaginal wall mechanical behavior. Elastase digestion induced marked changes in biaxial mechanical properties, suggesting that elastin may play an important role in vaginal wall mechanical function. Constitutive model evaluation resulted in the selection of a diagonal two-fiber family strain energy function and suggests that collagen fibers within the vaginal wall extracellular matrix (ECM) may be primarily oriented diagonally with a slight preference towards the circumferential direction. Further, our results suggest that elastin-collagen interactions may be important for vaginal wall homeostasis. The present findings may help to understand the underlying mechanisms of POP and aid in the development of growth and remodeling models for improved assessment and prediction of changes in structure-function relationships with prolapse development. / 0 / Katy Robison

Techn. Universiẗat, Diss., 2002--Braunschweig.

Elastin is a polymeric protein of the extracellular matrix that imparts the characteristics of extensibility and elastic recoil to tissues. Recombinant polypeptides based on the domain structures and sequences of human elastin self-assemble into organized fibrous structures, with physical properties similar to those of native polymeric elastin. Elastin self-assembly is initiated by a temperature-induced phase separation, called coacervation. Previous to this work, coacervation temperature had been the only parameter available to measure propensity for self-assembly. A variety of techniques were developed using spectrophotometry, microscopy, and rheometry to differentiate the stages of self-assembly, thereby enabling independent observation and quantitation of each stage, and allowing investigations into properties of polypeptides and solution conditions affecting these stages. Kinetic analysis of self-assembly yielded two additional parameters: coacervation velocity and maturation velocity. Examining the effects of agitation, salt concentration, temperature, polypeptide concentration, size of a polypeptide, hydrophobic domain sequence, and cross-linking domain structure on the kinetics demonstrated that coacervation and maturation are independent stages of self-assembly involving distinct mechanisms. Microscopic observations showed that protein-rich droplets of coacervate grew by coalescence to a stable droplet size, which correlated to differences in maturation velocities between polypeptides. Coacervate droplet growth appeared limited by the formation of organized polypeptide at the surface of the droplets, decreasing surface fluidity. Many of the general principles of the physical chemistry of colloids and emulsions appeared to apply to the formation, growth and stabilization of coacervates of the elastin-like polypeptides. Self-assembly in the presence of non-elastin, matrix-associated proteins showed that these proteins maintained the coacervate as small droplets, which sometimes flocculated into fibre-like structures. Rheometry demonstrated a second temperature-induced transition above the coacervation temperature, which resulted in gelation and viscoelastic characteristics similar to microgels. Together, these observations have resulted in a greater level of understanding of the entire self-assembly process, and provided a comprehensive model of elastin-like polypeptide self-assembly that relates to in vivo assembly of elastic fibres.

Elastin is a polymeric protein of the extracellular matrix that imparts the characteristics of extensibility and elastic recoil to tissues. Recombinant polypeptides based on the domain structures and sequences of human elastin self-assemble into organized fibrous structures, with physical properties similar to those of native polymeric elastin. Elastin self-assembly is initiated by a temperature-induced phase separation, called coacervation. Previous to this work, coacervation temperature had been the only parameter available to measure propensity for self-assembly. A variety of techniques were developed using spectrophotometry, microscopy, and rheometry to differentiate the stages of self-assembly, thereby enabling independent observation and quantitation of each stage, and allowing investigations into properties of polypeptides and solution conditions affecting these stages. Kinetic analysis of self-assembly yielded two additional parameters: coacervation velocity and maturation velocity. Examining the effects of agitation, salt concentration, temperature, polypeptide concentration, size of a polypeptide, hydrophobic domain sequence, and cross-linking domain structure on the kinetics demonstrated that coacervation and maturation are independent stages of self-assembly involving distinct mechanisms. Microscopic observations showed that protein-rich droplets of coacervate grew by coalescence to a stable droplet size, which correlated to differences in maturation velocities between polypeptides. Coacervate droplet growth appeared limited by the formation of organized polypeptide at the surface of the droplets, decreasing surface fluidity. Many of the general principles of the physical chemistry of colloids and emulsions appeared to apply to the formation, growth and stabilization of coacervates of the elastin-like polypeptides. Self-assembly in the presence of non-elastin, matrix-associated proteins showed that these proteins maintained the coacervate as small droplets, which sometimes flocculated into fibre-like structures. Rheometry demonstrated a second temperature-induced transition above the coacervation temperature, which resulted in gelation and viscoelastic characteristics similar to microgels. Together, these observations have resulted in a greater level of understanding of the entire self-assembly process, and provided a comprehensive model of elastin-like polypeptide self-assembly that relates to in vivo assembly of elastic fibres.

Besides collagen, elastin is the most common connective tissue structural protein in vertebrates and similar to collagen relatively resistant to non-specific degradation. Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases, the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases. Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with cathepsin V displaying the highest activity among all known mammalian elastases. Despite a shared amino acid sequence identity of over 80% between cathepsins V and L and very similar subsite specificities, only cathepsin V has a potent elastase activity whereas cathepsin L lacks it. A series of chimera mutants containing various proportions of cathepsin V and cathepsin L were constructed in an attempt to define a specific region needed for elastin degradation. It was found that retaining the peptide sequence region from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids 112-119) contributes approximately 60% of activity retention. Several additional mutant proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin L were constructed and evaluated for their elastolytic activities. The results obtained with those mutant cathepsin proteins support the importance of the amino acid region spanning the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V, this peptide region is located below subsite binding pocket S2 and forms a wall-like barrier which may act as an exosite for the productive binding of cross-linked elastin.