Abstract
Myofibroblasts are activated fibroblasts involved in tissue repair and cancer. They are characterized by de novo expression of α-smooth muscle actin (α-SMA), immunoregulatory phenotype and paracrine interaction with normal and tumorigenic cells leading to cell proliferation. At the end of wound-healing myofibroblasts undergo apoptotic cell death, whereas in vitro-activated fibroblasts are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response. Furthermore, myofibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this study, we generated and analysed clusters such as spheroids from human primary cutaneous myofibroblasts, which represent a part of stromal microenvironment better than established cell lines. Therefore, we evaluated apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation did not trigger apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α-SMA in protein extracts of spheroids, the cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts have undergone a deactivation process within spheroids. The cells of spheroids reverted to adhesion growth preserved their proliferation capability and can re-acquire a myofibroblastic phenotype. Moreover, the spontaneous formation of clusters on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and suggests that fibroblast clusters could be a cell reservoir regulating tissues turnover.
Highlights
Human fibroblasts represent a very heterogeneous cell population, that in adult body exhibits embryonic and functional diversities.[1]
The presence of intermediate filaments and stress fibres was evaluated in monolayer cells by vimentin immunofluorescence and phalloidin staining analysis, respectively (Figure 1a). This investigation detected the presence of vimentin intermediate filaments and stress fibres that are cytoskeletal markers of protomyofibroblasts, the cell type representing the intermediate step during fibroblasts to myofibroblasts differentiation process.[13]
Some authors consider neck skin fibroblasts originated from neural crest tissue, most fibroblasts are considered mesodermal cells.[2]
Summary
Human fibroblasts represent a very heterogeneous cell population, that in adult body exhibits embryonic and functional diversities.[1] most fibroblasts are considered mesodermal cells, some reports showed that fibroblasts of neck skin are derived from neural crest tissue.[2] Fibroblasts maintain the homoeostasis of the extracellular matrix (ECM), but can acquire an immunoregulatory phenotype.[2] it is known that activated fibroblasts produce large amounts of cyclooxygenase-2 (COX-2) and proinflammatory cytokines, the extent of fibroblast activation depends on the tissue type.[3] Activated fibroblasts in healing wounds, fibrotic or cancer tissue express de novo α-smooth muscle actin (α-SMA), show increased levels of growth factors secretion and ECM-degrading proteases: these processes are regulated by inflammation and are involved in the differentiation of fibroblasts into myofibroblasts.[2,4,5,6] At the end of wound healing, activated fibroblasts undergo apoptotic cell death, whereas in vitro fibroblasts endure a programmed necrosis-like cell death, called nemosis.[7,8] In particular, it is known that in vitro clusters of human dermal fibroblasts, named spheroids, are activated to produce massive amounts of COX-2, prostaglandins, proinflammatory cytokines and growth factors and thereby undergo nemosis associated with spheroids decomposition.[8] It is noteworthy that a recent study showed that nemosis is a reversible process.[9]
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