Abstract
Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development and homeostasis. However, during the formation of HS, whether and how autophagy is regulated in dermal fibroblasts are still far from elucidated. Here we detected the autophagic capacity in HS and normal skin (NS) counterparts, explored and verified the key regulatory molecules of autophagy in HS-derived fibroblasts (HSFs), and validated the data using rabbit ear scar model. Transmission electron microscopy (TEM) and immunostaining data showed that LC3-positive cells and autophagosomes in HS/HSFs were more intensive relative to those in NS/NSFs groups. Knockdown of LC3 (shLC3) could significantly block the expressionof type I collagen (Col 1, p < 0.01) and type III collagen (Col 3, p < 0.01) and thus inhibit the fibrosis of HSFs. shLC3 resistant to autophagy was shown to be Bcl-xL-, not Bcl-2-dependent, and silencing of Bcl-xL (sibcl-xL) significantly increased apoptosis of HSFs (p < 0.01). Immunofluorescence results showed that instead of inhibiting α-SMA protein expression, shLC3 could change its architecture arrangement in HSFs. sibcl-xL showed that Bcl-xL was a key signaling molecule involved in HSFs autophagy. More importantly, both shLC3 and sibcl-xL obviously improved the appearance and architecture of the rabbit ear scar, and reduced scar formation on the rabbit ear. Therefore, the aberration of LC3 protein processing compromised autophagy in HS might associate with its pathogenesis in wound repair. LC3 regulated HS fibrosis by controlling the expression of Bcl-xL in HSFs. Thus, Bcl-xL might serve as a potential molecular target, providing a novel strategy for HS therapy.
Highlights
Autophagy is used as a cellular response in which proteins, organelles, and portion of cytoplasm are engulfed, digested, and recycled to sustain cellular metabolism during stress [1, 2]
Immunocytochemistry data showed that microtubuleassociated protein light chain 3 (LC3) was localized in both normal skin (NS) and Hypertrophic scar (HS) (Figure 1C), its expression level was significantly higher in HS (Figure 1C)
Our results showed that in HS-derived fibroblasts (HSFs), α-SMA took the shape of fiber filaments in the control (AdNT and shNT, Figure 2E and 2F) and adenovirus vector for LC3B (AdLC3) (Figure 2E) groups, but in showed that knockdown of LC3 (shLC3) group, the structure of α-SMA was appeared as a tadpole and rod (Figure 2F)
Summary
Autophagy is used as a cellular response in which proteins, organelles, and portion of cytoplasm are engulfed, digested, and recycled to sustain cellular metabolism during stress [1, 2]. Prolonged autophagy activation can result in dysfunction of cellular organelles and even selfdestruction of cells [3, 4] This process is physiologically essential for the maintenance of cellular functions, cell viability, differentiation and development in mammals [58], and serves as an adaptive mechanism to protect organisms against diverse pathological insults [5, 9, 10]. Existing studies [23,24,25,26,27] suggest that autophagy in skin dermis is associated with the maintenance, viability, differentiation and survival of fibroblasts during wound healing and repair, so as to lead to the pathogenesis of pathological scars, such as HS and keloid
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