C.M. Kielty

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.

Fibrillin microfibrils are reduced in skin exhibiting striae distensae

Striae distensae (striae: stretch marks) are a common disfiguring condition associated with continuous and progressive stretching of the skin—as occurs during pregnancy. The pathogenesis of striae is unknown but probably relates to changes in those structures that provide skin with its tensile strength and elasticity. Such structures are components of the extracellular matrix, including fibrillin, elastin and collagens. Using a variety of histological techniques, we assessed the distribution of these extracellular matrix components in skin affected by striae. Pregnant women were assessed for the presence of striae, and punch biopsies were obtained from lesional striae and adjacent normal skin. Biopsies were processed for electron microscopy, light microscopy and immunohistochemistry. For histological examination, 7u2003μm frozen sections were stained so as to identify the elastic fibre network and glycosaminoglycans. Biopsies were also examined with a panel of polyclonal antibodies against collagens I and III, and fibrillin and elastin. Ultrastructural analysis revealed alterations in the appearance of skin affected by striae compared with that of normal skin in that the dermal matrix of striae was looser and more floccular. Light microscopy revealed an increase in glycosaminoglycan content in striae. Furthermore, the number of vertical fibrillin fibres subjacent to the dermal–epidermal junction (DEJ) and elastin fibres in the papillary dermis was significantly reduced in striae compared with normal skin. The orientation of elastin and fibrillin fibres in the deep dermis showed realignment in that the fibres ran parallel to the DEJ. However, no significant alterations were observed in any other extracellular matrix components. This study identifies a reorganization and diminution of the elastic fibre network of skin affected by striae. Continuous strain on the dermal extracellular matrix, as occurs during pregnancy, may remodel the elastic fibre network in susceptible individuals and manifest clinically as striae distensae.

Clinical features of photodamaged human skin are associated with a reduction in collagen VII

Chronically sun‐exposed or photodamaged human skin is characterized by a number of clinical features, including wrinkles. However, little is known about the molecular mechanisms that underlie these features. We investigated the hypothesis that the mechanism of wrinkle formation may involve loss of anchoring fibrils, composed mainly of collagen VII, which are important in maintaining dermal‐epidermal junction integrity. Ten volunteers with moderate to severe photodamage of dorsal forearm skin were recruited to the study. Using immunohistochemistry, transmission electron microscopy and in situ hybridization, we compared collagen VII protein and mRNA content of photodamaged forearm skin with that of sun‐protected hip and upper inner arm skin from the same subjects. Numbers of anchoring fibrils per linear μm of basement membrane (mean ± SEM) were significantly lower in photodamaged skin (1·79±0·10) as compared with sun‐protected hip (2·28±0·11) and upper inner arm skin (2·21±0·10) (P<0·01), and similarly keratinocyte expression of collagen VII mRNA, quantitated as number of positively stained keratinocytes per high power field, was significantly reduced in photodamaged skin (6·3±2·5) as compared with sunprotected hip (20·0±5·6) and upper inner arm skin (17·7±4·9) (P<0·001). Semiquantitative assessment of immunohistochemical staining for collagen VII showed a non‐significant reduction in photodamaged skin as compared with sun‐protected skin. We propose that reduced content of collagen VII in photodamaged skin contributes to wrinkle formation by weakening the bond between the dermis and epidermis.

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization

Fibrillin–containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal—epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal—epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal—epithelial junction is not well defined. We have used fresh tissues, freshlyisolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin‐1 and fibrillin‐2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co‐ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.

Distribution and expression of type VI collagen in photoaged skin

Backgroundu2003Several of the characteristic clinical features of photoaged skin, including wrinkling, are thought to be dependent on changes in the dermal matrix brought about by chronic sun exposure. Such changes include reductions in collagens I, III and VII, an increase in elastotic material in the reticular dermis and a marked reduction in the microfibrillar glycoprotein fibrillin.