Michael J. Sherratt

Molecular aspects of skin ageing

Ageing of human skin may result from both the passage of time (intrinsic ageing) and from cumulative exposure to external influences (extrinsic ageing) such as ultraviolet radiation (UVR) which promote wrinkle formation and loss of tissue elasticity. Whilst both ageing processes are associated with phenotypic changes in cutaneous cells, the major functional manifestations of ageing occur as a consequence of structural and compositional remodeling of normally long-lived dermal extracellular matrix proteins. This review briefly considers the effects of ageing on dermal collagens and proteoglycans before focusing on the mechanisms, functional consequences and treatment of elastic fibre remodeling in ageing skin. The early stages of photoageing are characterised by the differential degradation of elastic fibre proteins and whilst the activity of extracellular matrix proteases is increased in photoexposed skin, the substrate specificity of these enzymes is low. We have recently shown however, that isolated fibrillin microfibrils are susceptible to direct degradation by physiologically attainable doses of UV-B radiation and that elastic fibre proteins as a group are highly enriched in UV-absorbing amino acid residues. Functionally, elastic fibre remodeling events may adversely impact on: the mechanical properties of tissues, the recruitment and activation of immune cells, the expression of matrix metalloproteinases and cytokine signaling (by perturbing fibrillin microfibril sequestration of TGFβ). Finally, newly developed topical interventions appear to be capable of regenerating elements of the elastic fibre system in ageing skin, whilst systemic treatments may potentially prevent the pathological tissue remodeling events which occur in response to elastic fibre degradation.

Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling

Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.

Gastroduodenal dysfunction and bacterial colonisation of the ventilated lung

The source of ventilator-associated pneumonia (gastric or oropharyngeal flora) remains controversial. We investigated the source of bacterial colonisation of the ventilated lung in 100 consecutive intensive-care patients. Gram-negative bacilli were isolated from the lower respiratory tract in 19 patients. Bacteria isolated from the stomach contents either previously or at the same time were identical to lower respiratory isolates in 11 patients. No gram-negative oropharyngeal isolate was identical to a lower respiratory tract isolate. Gastric bacterial overgrowth with gram-negative bacilli was associated with the presence of bilirubin in the stomach contents. Detectable bilirubin was also associated with subsequent acquisition of gram-negative bacilli in the lower respiratory tract. Only 5 gastric aspirate specimens with pH < 3.5 contained gram-negative bacilli. These results establish a relation between duodenal reflux and subsequent bacterial colonisation of the lower respiratory tract. Restoration of normal gastroduodenal motility might help prevent pneumonia in intensive-care patients.

Tissue elasticity and the ageing elastic fibre

The ability of elastic tissues to deform under physiological forces and to subsequently release stored energy to drive passive recoil is vital to the function of many dynamic tissues. Within vertebrates, elastic fibres allow arteries and lungs to expand and contract, thus controlling variations in blood pressure and returning the pulmonary system to a resting state. Elastic fibres are composite structures composed of a cross-linked elastin core and an outer layer of fibrillin microfibrils. These two components perform distinct roles; elastin stores energy and drives passive recoil, whilst fibrillin microfibrils direct elastogenesis, mediate cell signalling, maintain tissue homeostasis via TGFβ sequestration and potentially act to reinforce the elastic fibre. In many tissues reduced elasticity, as a result of compromised elastic fibre function, becomes increasingly prevalent with age and contributes significantly to the burden of human morbidity and mortality. This review considers how the unique molecular structure, tissue distribution and longevity of elastic fibres pre-disposes these abundant extracellular matrix structures to the accumulation of damage in ageing dermal, pulmonary and vascular tissues. As compromised elasticity is a common feature of ageing dynamic tissues, the development of strategies to prevent, limit or reverse this loss of function will play a key role in reducing age-related morbidity and mortality.

Fibrillin microfibrils are stiff reinforcing fibres in compliant tissues.

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Youngs modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Youngs modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Characterizing the elastic properties of tissues

The quality of life of ageing populations is increasingly determined by age-related changes to the mechanical properties of numerous biological tissues. Degradation and mechanical failure of these tissues has a profound effect on human morbidity and mortality. Soft tissues have complex and intricate structures and, similar to engineering materials, their mechanical properties are controlled by their microstructure. Thus age-relate changes in mechanical behavior are determined by changes in the properties and relative quantities of microstructural tissue components. This review focuses on the cardiovascular system; it discusses the techniques used both in vivo and ex vivo to determine the age-related changes in the mechanical properties of arteries.

Self-assembly and gelation properties of α-helix versus β-sheet forming peptides

We have investigated the self-assembly and gelation properties of a set of four octa-peptides: AEAEAKAK, AEAKAEAK, FEFEFKFK and FEFKFEFK. The phenylalanine based peptides adopt β-sheet conformations in solution and the alanine based peptides form α-helices. No self-assembly in solution was observed for AEAKAEAK but AEAEAKAK was found to self-assemble forming thick, rigid fibres with a diameter of ∼6 nm. These fibres were composed of two fibrils aggregating side by side to form “pearl-necklace” morphologies. No gelation was observed for AEAEAKAK in the concentration range investigated (0 to 100 mg ml−1). In contrast, both phenylalanine based peptides were found to self-assemble in solution and to form hydrogels at an initial concentration of ∼8 mg ml−1. Similar morphologies were observed for both peptides corresponding to a relatively homogeneous dense network of semi-flexible fibres with a mesh size of ∼15 to 30 nm depending on the concentration. The fibre diameter was found to be ∼4 nm in good agreement with models found in the literature. TEM micrographs clearly showed that these fibres have a helicoidal or twisted structure. Comparison of TEM with AFM data highlighted the influence of substrate chemistry on the macromolecular assembly of small peptides. In contrast small angle neutron scattering (SANS) approaches, which allow for the probing of hydrogel morphology and structure without the need for sample preparation on solid substrates, provide vital data on hydrogel morphology in solution.

Fibrillin-rich microfibrils: elastic biopolymers of the extracellular matrix.

Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic ‘beads-on-a-strong’ appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.