Attenuation of Flightless I Increases Human Pericyte Proliferation, Migration and Angiogenic Functions and Improves Healing in Murine Diabetic Wounds

Abstract

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor β (PDGFRβ) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.

Multifunctional Roles of the Actin-Binding Protein Flightless I in Inflammation, Cancer and Wound Healing

“…Flii appears to impair angiogenesis in diabetic wounds with endothelial cells isolated from Flii overexpressing mice showing disrupted tight junction formation and reduced micro vessel sprouting (Ruzehaji et al, 2014). Diabetic patients, which display elevated Flii expression, also have a reduced number of pericytes, which work in tandem with endothelial cells to form stable, functional blood vessels (Thomas et al, 2020). Heterozygous knockout of Flii results in an upregulation of pro-angiogenic VEGF expression, and increased numbers of both endothelial cells and pericytes in diabetic mouse wounds (Ruzehaji et al, 2014;Thomas et al, 2020).…”

“…Diabetic patients, which display elevated Flii expression, also have a reduced number of pericytes, which work in tandem with endothelial cells to form stable, functional blood vessels (Thomas et al, 2020). Heterozygous knockout of Flii results in an upregulation of pro-angiogenic VEGF expression, and increased numbers of both endothelial cells and pericytes in diabetic mouse wounds (Ruzehaji et al, 2014;Thomas et al, 2020). In vitro, treatment with FnAb stimulates HUVEC cells to form capillary tubes and FnAb-containing matrigel plugs inserted under the skin of mice were found to have a fourfold increase in the length of functional vessels that contained erythrocytes (Ruzehaji et al, 2014).…”

Pro- and Anti-Angiogenic Factors: Their Relevance in Diabetic Foot Syndrome—A Review

“…Obviously, the higher presence of pericytes improved and hastened wound healing. 87 Though the issue requires further investigation, these studies show a detrimental effect of FLII on angiogenesis and wound healing of diabetic patients.…”

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/β-Catenin Signaling Pathway

“…Flii siRNA knockdown was performed in late-stage human cSCC cell line MET-1, which was originally collected from an invasive primary tumor from the forehead skin of an adult immunosuppressed patient and gifted by Professor Charlotte Proby from the University of Dundee [32]. Protocols used for the Flii knockdown were optimized as previously described [33]. Briefly, MET-1 cells were cultured in antibiotic-and serum-free MET-1 media overnight to synchronize the cell cycle.…”

“…Cell proliferation assays were performed on MET-1 cells following Flii siRNA knockdown according to the manufacturer's protocols (Roche Applied Science, Sydney, Australia) and previously described protocols [33]. Cells were counted using a hemocytometer, and 6 replicates of 5000 cells per well for each treatment were seeded in a well of a 96-well plate in serum free MET-1 media overnight to synchronize cell cycle.…”