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Title: Myofibroblasts and the pathogenesis of Dupuytren's disease
Author: Suleman Verjee, Liaquat
ISNI:       0000 0004 2695 8145
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Dupuytren’s disease is defined in Green’s Operative Hand Surgery as a condition of the hand characterised by the development of new tissue in the form of nodules and cords. Hand function can be significantly impaired and whilst the mainstay of treatment is surgery, recurrence has been reported in approximately 40-50% of patients. Myofibroblasts are central to the pathogenesis of Dupuytren’s disease although what regulates the myofibroblast phenotype during Dupuytren’s disease remains unclear. This thesis set out to test the hypothesis that the myofibroblast phenotype in Dupuytren’s disease is regulated by dermal fibroblasts and inflammatory mediators, specifically advanced glycation end products (AGEs) and TNF-α. I aimed to (1) systematically characterise the distribution of myofibroblasts (α-smooth muscle actin positive cells (α-SMA)) throughout excised Dupuytren’s tissue in relation to clinical disease activity (fixed flexion deformity and recurrence); (2) establish an in vitro contraction model to examine Dupuytren’s myofibroblasts and (3) use this model to examine how myofibroblast phenotype may be regulated by dermal fibroblasts and inflammatory mediators. I examined 103 Dupuytren’s cords and correlated the histological findings with the patient’s clinical flexion contracture and disease severity. Histological nodules, defined as a focal condensation of α-SMA positive cells, were seen in two-thirds of excised digital cord samples. These nodules were found in the vicinity of the proximal interphalangeal joints of the fingers. Nodules were more frequently observed in digits with less severe contractures and more active disease, whereas non-nodular cords were significantly associated with advanced digital contracture, reflecting end stage disease. Based on our current understanding of how myofibroblasts thrive on tension and contract against resistance, a mechanism for digital contraction has been proposed: nodules containing highly contractile myofibroblasts are present in active Dupuytren’s disease where digital flexion and extension of the interphalangeal joints is still possible and these nodules involute in end-stage disease, where severe contractures limit digital movement. In order to further study Dupuytren’s myofibroblasts, I also compared cell contraction in stress-released collagen gels with that in restrained gels using a culture force monitor (CFM). With the latter I was able to reliably measure cell-generated forces within restrained collagen gels. By using genetically matched sets of cells from patients, I also correlated cell contractility with α-SMA expression. Unlike control skin cells that initially contract and then plateau, Dupuytren’s myofibroblasts show continually increasing contractile force. Furthermore, increased contractility of Dupuytren’s myofibroblasts was reflected by higher α-SMA protein content and alignment of the α-SMA to stress fibres. Interestingly mRNA levels of α-SMA were similar in all matched cells, reflecting post-translational regulation of protein levels in myofibroblasts. Based on the observation that recurrence is significantly reduced following dermofasciectomy, I also tested whether dermal fibroblasts down-regulate the contractile Dupuytren’s myofibroblast. I co-cultured Dupuytren’s myofibroblasts and dermal fibroblasts and found that contractility of Dupuytren’s cells is not influenced by dermal cells. Therefore, reduced recurrence rate following dermofasciectomy may be due to other factors, such as more complete excision of affected tissue combined with reduced tension following application of full thickness skin grafts. Finally, recent studies have suggested that inflammatory mediators and AGE-RAGE interaction play a role in the development of the myofibroblast phenotype. I therefore used the CFM to examine the effect of AGEs and TNF-α on myofibroblast contractility. No difference in contractility of dermal fibroblasts was observed with AGEs, although a two-fold increase was seen with TNF-α. Overall, I have characterised the distribution of myofibroblasts in Dupuytren’s tissue and these data have led to interesting insights into disease pathogenesis. I have also established a reliable in vitro model for assessing Dupuytren’s myofibroblast phenotype and used this to demonstrate that whilst dermal fibroblasts do not downregulate myofibroblast activity, pro-inflammatory mediators may play a key role in modulating myofibroblast phenotype in this disease.
Supervisor: Brennan, Fionula ; Nanchahal, Jagdeep ; Midwood, Kim Sponsor: Healing Foundation ; British Society for Surgery of the Hand ; Hammersmith Hospitals Trustees' Research Committee ; Kennedy Institute of Rheumatology Trustees, Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral