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Impaired Wound Healing and Inflammation: The Role of the Dermal Fibroblast. Phenotypic Changes in the Human Dermal Fibroblast with Inflammation; Potential Impact on Wound Healing
Al-Rikabi, Aaiad H.A.
Al-Rikabi, Aaiad H.A.
Publication Date
2019
End of Embargo
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The University of Bradford theses are licenced under a Creative Commons Licence.
Peer-Reviewed
Open Access status
Accepted for publication
Institution
University of Bradford
Department
Faculty of Life Sciences. Centre of Skin Science
Awarded
2019
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Additional title
Abstract
Dermal fibroblasts positively contribute throughout the wounding response by
secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on
the functional, secretory, and molecular properties of these cells. This study
aims to understand how the effect of the pro-inflammatory cytokine TNF-α
modulates both healthy and diabetic dermal fibroblast phenotype.
To mimic a chronic inflammatory environment and assess whether fibroblasts
respond similarly in different anatomical sites, donor-matched fibroblasts from
face and scalp were pre-incubated for 3 days with different concentrations (2.5,
25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation
by day 14 in cells from both sites and stimulated (papillary) metabolic activity at
day 14.
However, this did not correlate with an increase in papillary cell senescence
since this did not appear until passage 17, and then only at a supra pathophysiological concentration.
Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α
significantly inhibited the cells migration, particularly in diabetic fibroblasts,
suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the
secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium
was collected to assess its effect on other dermal fibroblasts, however, this had
no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the
secretion of soluble paracrine factors that induce MMP activity in non-diabetic
fibroblasts, mirroring previous observations that a pro-inflammatory environment
can increase proteolytic activity, and indicating that diabetic fibroblasts were
again more sensitive than healthy. No difference was observed with MMP-9
activity and nor did the results with dermal fibroblasts reach statistical
significance, perhaps because of a relatively low n-number.
The ability of TNF-α to modulate the expression of genes associated with the
ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was
investigated. There was no change in Sirt1 and Sirt6 expression and no
evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α
significantly induced mRNA expression of MMP-1 in healthy non-scratched and
scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There
was also considerable donor variability that prevented statistical significance
being achieved under the other conditions.
The secretion of various cytokines associated with inflammation was compared
in healthy and diabetic fibroblasts in the presence and absence of TNF-α.
Seven cytokines were secreted, by healthy and diabetic male and female
fibroblasts, although diabetic female fibroblasts did not secrete two of them.
TNF-α stimulated secretion of cytokines in healthy and diabetic, male and
female cells but the profiles of those released were different between the
different groups. There was no TNF-α induced paracrine effect on cytokine
secretion by healthy dermal fibroblasts.
In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype.
Understanding these functional and molecular changes in response to
inflammatory cytokines will give a better understanding of the differences
between fibroblast activity in normal physiological wound healing and chronic or
diabetic non-healing wounds.
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Type
Thesis
Qualification name
PhD