Macrophage phenotype is determinant for fibrosis development in keloid disease.

Keloid refers to a fibroproliferative disorder characterized by an accumulation of extracellular matrix (ECM) components at the dermis level, overgrowth beyond initial wound, and formation of tumor-like nodule areas. Treating keloid is still an unmet clinical need and the lack of an efficient therapy is clearly related to limited knowledge about keloid etiology, despite the growing interest of the scientific community in this pathology. In past decades, keloids were often studied in vitro through the sole prism of fibroblasts considered as the major effector of ECM deposition. Nevertheless, development of keloids results from cross-interactions of keloid fibroblasts (KFs) and their surrounding microenvironment, including immune cells such as macrophages. Our study aimed to evaluate the effect of M1 and M2 monocyte-derived macrophages on KFs in vitro. We focused on the effects of the macrophage secretome on fibrosis-related criteria in KFs, including proliferation, migration, differentiation, and ECM synthesis. First, we demonstrated that M2-like macrophages enhanced the fibrogenic profile of KFs in culture. Then, we surprisingly founded that M1-like macrophages can have an anti-fibrogenic effect on KFs, even in a pro-fibrotic environment. These results demonstrate, for the first time, that M1 and M2 macrophage subsets differentially impact the fibrotic fate of KFs in vitro, and suggest that restoring the M1/M2 balance to favor M1 in keloids could be an efficient therapeutic lever to prevent or treat keloid fibrosis.

Is Spheroid a Relevant Model to Address Fibrogenesis in Keloid Research?

Keloid refers to a fibro-proliferative disorder characterized by an accumulation of extracellular matrix at the dermis level, overgrowing beyond the initial wound and forming tumor-like nodule areas. The absence of treatment for keloid is clearly related to limited knowledge about keloid etiology. In vitro, keloids were classically studied through fibroblasts monolayer culture, far from keloid in vivo complexity. Today, cell aggregates cultured as 3D spheroid have gained in popularity as new tools to mimic tissue in vitro. However, no previously published works on spheroids have specifically focused on keloids yet. Thus, we hypothesized that spheroids made of keloid fibroblasts (KFs) could be used to model fibrogenesis in vitro. Our objective was to qualify spheroids made from KFs and cultured in a basal or pro-fibrotic environment (+TGF-ß1). As major parameters for fibrogenesis assessment, we evaluated apoptosis, myofibroblast differentiation and response to TGF-ß1, extracellular matrix (ECM) synthesis, and ECM-related genes regulation in KFs spheroids. We surprisingly observed that fibrogenic features of KFs are strongly downregulated when cells are cultured in 3D. In conclusion, we believe that spheroid is not the most appropriate model to address fibrogenesis in keloid, but it constitutes an efficient model to study the deactivation of fibrotic cells.

Beneficial Effects of a Blended Fibroin/Aloe Gel Extract Film on the Biomolecular Mechanism(s) via the MAPK/ERK Pathway Relating to Diabetic Wound Healing.

In diabetic patients, the process of wound healing is usually delayed or impaired. A diabetic environment could be associated with dermal fibroblast dysfunction, reduced angiogenesis, the release of excessive proinflammatory cytokines, and senescence features. Alternative therapeutic treatments using natural products are highly demanded for their high potential of bioactive activity in skin repair. Two natural extracts were combined to develop fibroin/aloe gel wound dressing. Our previous studies revealed that the prepared film enhances the healing rate of diabetic foot ulcers (DFUs). Moreover, we aimed to explore its biological effects and underlying biomolecular mechanisms on normal dermal, diabetic dermal, and diabetic wound fibroblasts. Cell culture experiments showed that the γ-irradiated blended fibroin/aloe gel extract film promotes skin wound healing by enhancing cell proliferation and migration, vascular epidermal growth factor (VEGF) secretion, and cell senescence prevention. Its action was mainly linked to the activation of the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway known to regulate various cellular activities, including proliferation. Therefore, the findings of this study confirm and support our previous data. The blended fibroin/aloe gel extract film displays a biological behavior with favorable properties for delayed wound healing and can be considered as a promising therapeutic approach in the treatment of diabetic nonhealing ulcers.

Halofuginone regulates keloid fibroblast fibrotic response to TGF-ß induction.

Keloids are characterized by increased deposition of fibrous tissue in the skin and subcutaneous tissue following an abnormal wound healing process. Although keloid etiology is yet to be fully understood, fibroblasts are known to be key players in its development. Here we analyze the antifibrotic mechanisms of Halofuginone (HF), a drug reportedly able to inhibit the TGF-ß1-Smad3 pathway and to attenuate collagen synthesis, in an in-vitro keloid model using patient-derived Keloid Fibroblasts (KFs) isolated from fibrotic tissue collected during the "Scar Wars" clinical study (NCT NCT03312166). TGF-ß1 was used as a pro-fibrotic agent to stimulate fibroblasts response under HF treatment. The fibrotic related properties of KFs, including survival, migration, proliferation, myofibroblasts conversion, ECM synthesis and remodeling, were investigated in 2D and 3D cultures. HF at 50 nM concentration impaired KFs proliferation, and decreased TGF-ß1-induced expression of α-SMA and type I procollagen production. HF treatment also reduced KFs migration, prevented matrix contraction and increased the metallo-proteases/inhibitors (MMP/TIMP) ratio. Overall, HF elicits an anti-fibrotic contrasting the TGF-ß1 stimulation of KFs, thus supporting its therapeutic use for keloid prevention and management.