We set out to examine the pathophysiological mechanisms of fibrosis in diffuse systemic sclerosis (SSc) using a tissue engineering approach. Skin fibroblasts were isolated from lesional skin of SSc patients with a disease duration of less than 1 year (early-stage SSc) or more than 10 years (late-stage SSc). Fibroblasts were also isolated from non-lesional skin and compared with normal fibroblasts isolated from healthy adults. Cells were cultured using a tissue engineering method to reconstruct a human dermis, and histologically observed. Dermal thickness was measured, as it reflects the global and intrinsic capacity of cells to reconstitute matrix. Collagen I, MMP-1, and MMP activity were evaluated. Cells were treated with TGFbeta1 or CTGF during dermis formation to study their fibrogenic role. Clinical severity of skin involvement was measured by a modified Rodnan score. Thickness of the dermis generated with non-lesional early-stage SSc fibroblasts was similar to normal cells. In contrast, reconstructed dermis from lesional early-stage SSc fibroblasts and non-lesional late-stage SSc cells was thinner, while lesional late-stage SSc fibroblasts made a thicker dermis. Dermis was always thicker when produced with TGFbeta1-treated cells, except when lesional late-stage SSc fibroblasts from patients with high Rodnan skin scores were used. CTGF did not affect dermal thickness. Measurements of collagen I and collagenases in the culture medium of the various reconstructed dermis could explain some of the changes observed. We conclude that the fibrotic phenotype of SSc fibroblasts varies with disease duration and with severity of skin involvement, and this is clearly visualized during in vitro dermis reconstruction.

Our hypothesis is that the development of lesional areas of skin in patients with systemic sclerosis (SSc) originates from the selection of profibrotic cell subpopulations within their non-lesional skin areas, due to their greater resistance to apoptosis. Sensitivity to apoptosis of early-stage or late-stage SSc fibroblasts as well as of healthy cells was compared using extrinsic or intrinsic apoptotic pathway-inducers. Subpopulations of non-lesional SSc cells and healthy cells obtained after repeated Fas-induced apoptosis were compared with respect to their fibrotic parameters such as collagen and MMP secretion. Only late-stage lesional SSc cells were more resistant to Fas-induced apoptosis than their non-lesional counterparts isolated from the same patient. This result correlated with an increase in the levels of the anti-apoptotic proteins cFLIPs and cIAP in lesional cells compared to non-lesional cells. Healthy and non-lesional cell populations could be selected to generate a subpopulation that was more resistant to apoptosis. However, only the late-stage non-lesional SSc fibroblast populations showed a significant decrease in MMP secretion, one of parameters of the fibrosis. Our results show that resistance to apoptosis is an important characteristic of the late-stage lesional SSc fibroblast phenotype. We thus hypothesized that a selection of specific fibroblast subpopulations from late-stage non-lesional SSc skin areas could be at the origin of lesional populations. These cells should become independent of any exogenous stimuli and can induce or maintain SSc skin lesions.

Scleroderma is a connective tissue disease characterized by skin and internal organs fibrosis, vasculopathy of small arteries and activation of the immune system. The objective of this project is to study the capacity of scleroderma fibroblasts to induce fibrosis using a model of tissue engineered dermis. Fibroblasts were isolated from skin biopsies of patient with scleroderma for less than one year (early stage) or for more than ten years (late stage). For each patient, two biopsies were taken, one from affected area and the other one from a non‐affected area. Control fibroblasts were obtained from healthy donors. Only fibroblasts isolated from the skin biopsies from affected area of patient at late stage of scleroderma were able to reconstruct dermis thicker than that obtained with control fibroblasts. All of the other fibroblasts tested formed dermis thinner than the one obtained with control fibroblasts. These observations are not the consequence of a change in the amount of fibroblast present, but due to a different turnover of extracellular matrix (matrix metalloproteinase‐1/collagen I) according to the stage and the area. These results suggest the necessary presence of exogenous factor(s) to induce fibrosis in the early stage of the disease, while in late stage these factor(s) would not be essential. This study was supported by CIHR, by the “Fondation de l’Hôpital St‐Sacrement” and a scholarship from FRSQ (VM).

Systemic sclerosis or scleroderma (SSc), is an autoimmune disease with an incidence of 20 people per million and per year in USA. No cure for SSc is currently available so, after a debilitating phase, death is frequently the outcome of this disease with a higher risk of mortality among Afro‐Americans and women. Causes of SSc are unknown for now but various factors seem to be involved like genetics or exposure to chemical or virus. One of the features of this disease is extracellular matrix excessive deposition which affect not only skin but also internal organs. Despite heterogeneity of cellular and molecular responses of fibroblasts (Fb), a larger proportion of SSc Fb express higher level of collagen than in normal ones. So, many groups postulated that SSc lesions could result from clonal expansion of a Fb sub‐population with a higher capacity to proliferate and/or a better resistance to apoptosis. Dermal Fb populations from 17 French‐Canadian volunteers (4 healthy and 13 SSc affected) were tested for proliferation and apoptosis parameters. We have found that SSc Fb proliferate with a lower rate than the normal one, excluding proliferation as a clonal selection origin. Nevertheless lesional SSc Fb were less sensitive than normal Fb or non‐lesional SSc Fb to Fas pathway apoptosis allowing to hypothesize that clonal selection could be performed via apoptotic pathway. This work was supported by grants from CHIR and FRSQ.