Enhanced galectin-7 expression favors wound healing

Abstract

Upon disruption of the skin barrier, a synchronized sequence of precise steps involving multiple cell types is activated to achieve wound healing. Though healing restores the physical barrier function, wound repair in adults -unlike in fetal life- frequently results in a non-physiological fibrotic scar. Unraveling the mechanisms underlying scarless wound healing would have broad clinical and cosmetical implications [1].Along the different stages of wound healing process (i.e.: hemostasis, inflammation, cell proliferation and re-epithelialization) several molecular mediators contribute to tissue repair programs. Among them, galectins (Gals) -a family of glycan-binding proteins that specifically recognize glyco-conjugates (N-acetyl-lactosamine disaccharide)- shape cellular circuitries in immune cells, endothelial cells, and fibroblasts impacting on broad spectrum of biological processes [2]. In particular, while the ubiquitously expressed Gal-1, Gal-3 or Gal-9 have proved to orchestrate immune response circuits, regulate angiogenic programs and impact on epithelial cell biology [2], Gal-7 may contribute to different events associated with the differentiation and development of stratified epithelia where it is preferentially expressed [3]. Modulated by several environmental stressors (UV irradiation, inflammation or chemical mutagenic stimuli), Gal-7 expression has been shown to control UVB-induced apoptosis in keratinocytes [3].In view of their ability to regulate cell-cell and cell-extracellular matrix interactions and to impact on immune and vascular programs, galectins raise as clear candidates to participate in the different stages of wound healing process. Indeed, a role for Gal-3 and Gal-7 but not for Gal-1 in corneal wound-healing, and for Gal-2 and 4 in re-epithelialization of intestinal wounds was suggested [4]. Also, altered epidermal early response (24-48hs) to UVB irradiation or to superficial injury -affecting exclusively epidermal layer- was observed in the absence of this lectin in mice [5], and reduced expression of Gal-7 was evidenced in sera and skin from patients with hypertrophic scars [6] and in lesional skin and sera from systemic sclerosis patients [7]. However, considering the broad spectrum of galectins´ biological functions, their different tissue/cell expression pattern and the complexity of the wound healing process, a better understanding of their role during skin regeneration should be further addressed.Herein, we first investigated changes in galectins´ expression levels upon skin wounding by analysing single-cell mRNA sequencing (scRNAseq) data from mice back skin samples isolated at 4 days after the introduction of 6-mm wounds, corresponding to a stage of active re-epithelialization [8]. Compared with unwounded tissue, a significant upregulation of Lgals7 expression in keratinocytes (basal epidermal, proliferating epidermal, spinous epidermal and hair follicle stem cells; Fig. 1A) and of Lgals1 expression in immune cell populations (macrophages, fibroblasts, T cells and dendritic cells, Fig. 1A), but not of other galectins, was observed. Thus, exclusively Lgals1 and Lgals7 expressed in immune and epithelial cells, respectively, could be part of the healing process which is in full development at day 4, as shown by the enhanced expression of cytokines (IL-1b, IL-10 and TNF-) and growth factors (Vegfa, Pdgfa and FGF7) intimately related with skin regeneration (Fig. 1B). Given that Lgals7 was found specifically upregulated in keratinocytes during murine wound healing, we then investigated the role of this lectin in maintenance of skin integrity in C57BL/6j WT mice compared to genetically engineered strains that either do not express Gal-7 (Lgals7-/-) or over-express it under keratin 14 promoter (Tg46) [5,9]. Though genetic engineered animals did not display phenotypical abnormalities, Lgals7-/- basal keratinocytes showed lower proliferation rate than those from WT mice (Fig 1B). Interestingly, 12hs after insulting (scratching), Tg46 animals showed a smaller area of injured ear skin compared to WT mice, and these last evidenced smaller injured area than Lgals7-/-mice, in toluidine blue assay (Fig 1C), suggesting that higher level of Gal-7 favours recovering of skin integrity. To further study the impact of endogenous Gal-7 in skin regeneration, 5mm2 wounds were performed in mice shaved back-skin, and wound area was daily monitored. Notably, healing process in Tg46 mice was faster than in WT and Lgals7-/- mice, evidenced by reduced mean wound area at days 3 and 6 post injury (Fig 1D). At day 3, injured areas in Tg46 mice were almost completely dry and crusted, while most of these areas in Lgals7-/-group were still wet. In contrast to previous findings [9], histopathological examination at day 6 showed an already achieved organized structure of dermal and epithelial layers in Tg46 mice compared to a still in process in WT group. Mice lacking Gal-7 showed poor dermis and epidermis restructuring. Differences in type of insult, site of application and temporal approach (single end point vs kinetics of the response) may explain dissimilitude to previous works.In conclusion, analysis of scRNAseq data and experiments in genetically-engineered mice models support a role for Gal-7 in skin regeneration and maintenance of homeostasis. Enhancement of Gal-7 expression upon environmental insults may represent a homeostatic response towards wound healing and restoration of skin physiology. Though further studies are required to reveal the specific underlying molecular mechanisms, our work identifies Gal-7 as a local checkpoint that controls skin homeostasis, and highlights its potential therapeutic relevance in skin regeneration disorders which constitute a serious medical problem.