The perivascular adventitia (PA) senses and responds to injuries in blood vessels and the tissues they feed. Cells in the PA form the outermost vascular layer, joining the circulatory system to other organs. Housing hematopoietic, mesenchymal and neuronal cells allows flexible adventitial responses to diverse perturbations. However, the PA response can also be pathogenic. Thickening of the adventitia may drive ischemia and hypertension. It can also be a niche for local lymphocyte priming in diseases such as idiopathic pulmonary arterial hypertension. Despite their importance, PA contributions to skin diseases were understudied. The hypothesis that contrasting two cutaneous diseases, scleroderma and discoid lupus erythematosus (DLE), would illuminate discrete PA alterations was explored. Vascular changes are prominent, but distinct, in both diseases. Studying perivascular adventitial changes in these diseases may yield insights into both dermal and vascular pathologies. PA fibroblasts in healthy human skin were phenotypically distinct from the surrounding dermal fibroblasts. In both scleroderma and DLE, PA fibroblasts expanded and expressed surface markers not observed in healthy skin including vascular cell adhesion molecule 1 (VCAM1), podoplanin (PDPN) and the p75 low affinity nerve growth factor receptor (NGFR). Elaborated networks of PA fibroblasts in DLE expressed VCAM1 and enmeshed dense, T cell-rich infiltrates. Transcriptional analyses indicated positive correlations between VCAM1, T cell chemoattractants and interleukin (IL)-15, which promotes their survival. Activated PA fibroblasts in DLE likely create a supportive niche for T cells infiltrating the skin. In contrast, enlarged PA fibroblast networks in scleroderma expressed NGFR in the absence of leukocyte infiltrates. This PA fibroblast phenotype was shared among reparative and pathologic scarring, and four dermal tumors. NGFR is a mesenchymal stem cell (MSC) marker, and expanded NGFR+ mesenchymal cells were immediately adjacent to cluster of differentiation (CD)34+ and CD73+ PA MSC. Expression of NGFR by PA fibroblasts is likely associated with reparative responses. Different stimuli induced VCAM1 and NGFR on cultured human dermal fibroblasts, supporting these as discrete activation states. In conclusion, these studies demonstrated the responsive and plastic nature of human dermal PA mesenchymal cells, and pointed to connections with vascular alterations in skin diseases.

Missing page 192. Page 194 was repeated, therefore one was omitted. / <p> The synthesis and degradation of proteins were studied during aging of cultured human fibroblasts. Equations were derived to yield expressions for the rates of protein degradation, export, and synthesis during exponential growth and steady state from the approach to equilibrium method of radioactively labeling protein. Old cells (cells from normal donors at late passage, cells from old donors, or cells from subjects which the accelerated aging phenotypes of Hutchinson-Gilford (progeria) and Werner syndromes) have a reduced growth rate (0.3-1.3%/hour) when cultured at low density compared to young cells (early-passage cells from normal donors) (2.0-2.5%/hour). Prior to the terminal passage in old cultures, this reduction in growth rate is related primarily to an increased rate of protein degradation (0.96-1.3%/hour in old cells compared to less than 0.55%/hour in young cells). Early-passage cells achieve rapid growth in low density cultures by increasing the protein synthetic rate and decreasing the degradation rate. In high density cultures where the net growth rate was close to zero, the rates of degradation and synthesis were similar in young and old cells prior to their terminal passage (1.9-2.5%/hour). In all cases the rate of protein export was small (less than 0.5%/hour) compared to the rate of protein synthesis. </p> Proteins synthesized by young and old cells were analyzed by two-dimensional gel electrophoresis and were found to be essentially identical in molecular weight and isoelectric points. Induction of synthesis of aberrant proteins by histidine starvation in the presence of histidinol did not reveal differences between early- and late-passage cells from young or old normal donors or from subjects with progeria or Wener Syndrome. Furthermore, there was no correlation between in vitro lifespan and the synthesis of aberrant protein. </p> <p> It is concluded that the increased degradation of proteins and the slow net growth of old cells and the reduced lifespan of cells from old normal donors and subjects with progeria or Werner Syndrome are not due to abnormal protein synthesis. This is contrary to the predictions of the error catastrophe theory of aging. </p> <p> The aberrant proteins synthesized during amino acid starvation are believed to result from amino acid substitution. Several observations reported here are consistent with this hypothesis: (i) No turnover of either native or substituted actins synthesized during histidine starvation of cultured human cells was; (ii) Changes in the isoelectric points of native and substituted actins are predicted by analyses based on the presumed changes in their amino acid composition; (iii) Estimates of the protein synthetic error rates during normal protein synthesis can be derived from a computer model of mRNA translation based on the proposed mechanism of mistranslation; these estimates are consistent under a variety of starvation conditions and are close to other estimates obtained independently for the error frequency in mammalian cells. </p> <p> In both young and old cultured human fibroblasts the error frquency at the histidine codon was calculated to be 1.1 ± 0.1 x 10⁻⁴ (mean+S.E.). Three lines of Sv40-transformed human fibroblasts had error frequencies 2-5 fold greater than their untransformed counterparts. Studies with a variety of other human and non-human cell types did not support the conclusion that transformation in general increased in rate of mistranslation. The observation of increased error frequencies in SV40-transformed human cells may be restricted to this viral transformation. </p> <p> The computer simulations of mRNA translation have provided a means of extrapolating error frequencies determined during amino acid starvation to the error frequency during normal protein synthesis. This model is of great interest for its potential use as a method of rapidly quantifying protein synthetic error frequencies in cultured cells. </p> / Thesis / Doctor of Philosophy (PhD)

Background: Periodontitis is an inflammatory condition rendering in degradation of tooth supporting tissue. In the inflammatory process cytokines, amongst others TNF-a, IL-1b and IL-6 play an important role in regulating the immune response. Periodontal Ehlers Danlos syndrome (pEDS) is a rare connective tissue disorder characterized by distinct oral manifestations such as periodontitis. Studies has shown that individuals with pEDS exhibit a mutation in the complement factors C1r and C1s. Aim: The aim of this study was to analyze the gene expression of C1r and C1s in human gingival fibroblasts and to investigate if the expression of these genes’ changes during inflammatory conditions. Methods: Human gingival fibroblasts (hGFBs) were cultured with TNF-a or IL-1b for 24h. RNA isolation, extraction and complementary DNA synthesis was made at the timepoint of 6h and 24h. An qRT-PCR analyses of the genes C1r, C1s and IL-6 were performed and the relative gene expression of C1r, C1s and IL-6 were calculated. Results: The expression of C1r and C1s increased after 24h in TNF-a and IL-1b compared to control by measuring the relative fold change of the genes by culturing fibroblast over time. The result was taken from three parallel samples and the result was significant. Conclusion: The findings point to a link between the inflammatory classical complement pathway and the connective tissue homeostasis. Hopefully, our findings will be one piece of the puzzle regarding the molecular events rendering in pEDS and thereby a potential treatment target.

While the stress response represents an example of allostasis that enables the organism to cope with environmental and psychosocial challenges, its chronic activation imposes an allostatic load that contributes to the cumulative wear and tear of the system and induces negative mental and physical health outcomes. Nonetheless, the underlying basis of the stress-disease connection is still poorly understood and represents a gap in the knowledge that requires further research. We investigated the effects of chronic glucocorticoid stimulation in three independent human primary fibroblast lines, as an in vitro model of chronic psychosocial stress. By deploying a longitudinal, high-frequency, repeated-measures strategy across their entire lifespan, we were able to determine that chronically stressed cells present a significant increase in their total energy expenditure and that this stress-induced hypermetabolism is linked to an acceleration of their biological aging. Expanding from our results and placing emphasis on the energetic costs associated with the activation of the stress response, we proposed the “Energetic Model of Allostatic Load”. This model proposes that chronic stress causes a redirection of the energetic resources towards allostatic responses and away from growth, maintenance, and repair processes, which in turn leads to the accumulation of damage that will further contribute to the development of disease and increased risk of mortality. Finally, we highlighted new avenues to quantify allostatic load and its link to health via the integration of systemic and cellular energy expenditure measurements together with classic biomarkers, that could contribute to further advances in the stress field.

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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<p>An essential stage of repair for a healing wound is the proliferation of cells in the damaged space. Cells such as fibroblasts, grow and migrate to aid in construction of new tissue and to close the wound. Current methods of studying fibroblast proliferation in wound healing include a 2D wound healing assay in which a cell monolayer is scratched, and the cells migrate into the pseudo-wound. However, this lacks the 3D architecture of a physiological wound. Current 3D models of wound healing often rely on the use of a preexisting matrix for structural assistance, however an isolated system of cell growth without requirement of structural aid may gather new insights on intercellular behavior and mechanical properties. Additionally, we to desire to fabricate a high through-put and easy to use 3D wound healing model than currently offered. Our engineering objective is to create a novel 3D model of wound healing.</p> <p><br></p> <p>This project aims to optimize fibroblast adhesion and proliferation for 3D microtissue fabrication by altering surface and extracellular matrix (ECM) properties to SU-8 scaffolding. Additionally, we consider the effect of different geometries on cell proliferation and cellular stresses/strains, fibronectin production as pseudo-wounds close, and make comparisons to intercellular cancer behavior. Our results show around a 66% decrease in overall culture time required for the microtissues to reach full confluency. Varying geometries in the tessellated design have revealed structural changes in the actin cytoskeleton formation of fibroblasts, and increased fibronectin production along edges of tensioned cells preparing to “close” the wound. When compared to human breast cancer cells, the cancer cells lack the ability to make critical cell to cell junctions that we observe in fibroblasts, noting the characteristic that cancer is like a wound that never heals.</p>