Fibrillin is an elastomeric extracellular protein within skin that may catalyse elastosis in chronic photodamage. It assembles into beaded-microfibrils that are structural but which also regulate cytokine signalling within the extracellular space and their specific loss from the papillary dermis is a biomarker of early photodamage. However, the impact of the intrinsic ageing process upon their structure and function is poorly defined. These studies, herein, investigate the intrinsic ageing of fibrillin-rich microfibrils and the downstream effects of the interaction of photoprotected and photodamaged microfibrils on the dermal fibroblast transcriptome. Methods Fibrillin abundance in young and old photoprotected and photoexposed skin was quantified. The ultrastructure of microfibrils extracted from young and old photoprotected skin and from matrices of in vitro cultured primary dermal fibroblasts was analysed by atomic force microscopy. Since fibrillin co-purifies with collagen VI microfibrils that share similar biochemical cell-interaction properties, alternative protein purification methods were explored. Finally, transcriptomic responses of primary dermal fibroblasts to irradiated and unirradiated purified exogenous fibrillin-rich microfibrils were investigated by RNA-sequencing. Validation of gene expression was performed at the protein level by ELISA. Results Fibrillin abundance, alongside microfibril periodicity and bead height, were reduced in aged photoprotected skin suggesting that these assemblies undergo intrinsic ageing. Newly synthesised microfibrils by fibroblasts from aged skin also exhibited reduced bead periodicity. However, the bead height of de novo microfibrils from fibroblast matrices, irrespective of the age of the sample donor, was similar to the population extracted from aged photoprotected skin. Investigations into new methods of purifying fibrillin discovered that immunoprecipitation of collagen VI also depleted fibrillin, suggesting a potential novel protein interaction. Decreases in RINT1, TP53, and an increase in Wnt5a gene expression, as part of the fibroblast transcriptomic response to unirradiated microfibrils, suggest fibrillin may compromise genomic stability. Exposure of fibroblasts to irradiated microfibrils upregulated CCL5 expression, a cytokine involved in inflammation. Validation at the protein level of this and other proinflammatory cytokines, however, found no correlation with gene expression data. Conclusions These data suggest that fibrillin-rich microfibrils are modified in intrinsic ageing. Unirradiated and irradiated microfibrils may orchestrate multiple cell-signalling pathways that underscore the molecular mechanisms of ageing. These findings may also have implications for the ageing phenotypes of other elastic fibre-rich organ systems.
|Date of Award||1 Aug 2019|
- The University of Manchester
|Supervisor||Michael Sherratt (Supervisor) & Rachel Watson (Supervisor)|
The intrinsic ageing of cutaneous fibrillin-rich microfibrils and the impact of photodamage upon their role as biosignalling molecules.
Mellody, K. (Author). 1 Aug 2019
Student thesis: Phd