Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.

The advanced glycation end products (AGEs) of proteins are common factors in the pathophysiology of a number of disorders related to aging. The skin generation of AGEs occurs mainly through nonenzymatic glycation reactions of extracellular matrix (ECM) proteins in the dermis. The AGEs have been touted as one of the factors responsible for healing impairment and loss of elasticity of healing skin, affecting growth, differentiation, and cellular motility, as well as cytokines response, metalloproteinases expression, and vascular hemostasis. In this study, we generated an in vitro full-thickness reconstructed skin based on a glycated collagen matrix dermal compartment to evaluate the effects of glycation on dermal ECM and ultimately on the epidermis. Epidermal differentiation and stratification patterns and the glycation-induced ECM changes were evaluated by histology, immunohistochemistry, and mRNA levels. In this study, we reported for the first time that changes in the dermal matrix caused by collagen I in vitro glycation processes also affect the epidermal compartment. We demonstrated that glycation of collagen induces expression of carboxymethyllysine in dermal and epidermal compartments and, consequently, an aging phenotype consisting of poor stratification of epidermal layers and vacuolization of keratinocyte cytoplasm. Increased expression of cell-cell adhesion markers, such as desmoglein and E-cadherin in glycated skins, is observed in the stratum spinosum, as well as an increased compression of dermal collagen matrix. We also submitted our 3D model of reconstructed glycated skin to screening of anti-AGE molecules, such as aminoguanidine, which prevented the glycated morphological status. Controlled human studies investigating the effects of anti-AGE strategies against skin aging are largely missing. In this context, we proposed the use of skin equivalents as an efficient model to investigate cellular interactions and ECM changes in the aging skin, and to elucidate the role of anti-AGEs molecules in this process.

Brain injuries such as trauma and stroke lead to glial scar formation by reactive astrocytes which produce and secret axonal outgrowth inhibitors. Chondroitin sulfate proteoglycans (CSPG) constitute a well-known class of extracellular matrix molecules produced at the glial scar and cause growth cone collapse. The CSPG glycosaminoglycan side chains composed of chondroitin sulfate (CS) are responsible for its inhibitory activity on neurite outgrowth and are dependent on RhoA activation. Here, we hypothesize that CSPG also impairs neural stem cell migration inhibiting their penetration into an injury site. We show that DCX+ neuroblasts do not penetrate a CSPG-rich injured area probably due to Nogo receptor activation and RhoA/ROCK signaling pathway as we demonstrate in vitro with neural stem cells cultured as neurospheres and pull-down for RhoA. Furthermore, CS-impaired cell migration in vitro induced the formation of large mature adhesions and altered cell protrusion dynamics. ROCK inhibition restored migration in vitro as well as decreased adhesion size.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-017-0565-8) contains supplementary material, which is available to authorized users.

Deficient wound healing is a common multifactorial complication in diabetic patients, but the cellular and molecular mechanisms involved are poorly defined. In the present study, we analyzed the effects of hyperglycemia on integrins expression in rat dermal fibroblasts and addressed its role in cell adhesion and migration. Diabetes Mellitus was induced in rats by streptozotocin injection and maintained for 30 days. Primary cultures of dermal fibroblasts from control and diabetic rats were maintained under low glucose (5 mM D-glucose) or high glucose (30 mM D-glucose) for 7 days. Cell adhesion and migration were studied by kymography, transwell, and time-lapse assays, and the expressions of integrin subunits αv and α5 were studied by immunocytochemistry and western blotting. Fibroblasts derived from diabetic rats confirmed a reduced migration speed and delayed spreading compared to fibroblasts derived from control rats. The membrane fraction of diabetic-derived fibroblasts showed a decrease of integrin subunits α5 and αv, which was confirmed by immunocytochemistry assays. A reduction in the pericellular fibronectin matrix was also observed. The exposure of diabetic-derived cells to a higher concentration of exogenous fibronectin improved migration velocity and the expression of αv but did not completely restore their migration capacity. In conclusion, the mechanisms involved in the deleterious effects of Diabetes Mellitus on wound healing include the ability of fibroblasts to secrete and to adhere to fibronectin.

Crotoxin (CTX) is the primary toxin of South American rattlesnake Crotalus durissus terrificus venom. CTX reduces tumour mass, and tumour cell proliferation and these effects seem to involve the formation of new vessels. Angiogenesis has a key role in tumour growth and progression and is regulated by macrophage secretory activity. Herein, the effect of CTX on macrophage secretory activity associated with angiogenesis was investigated in vitro. Thymic endothelial cells (EC) were incubated in the presence of macrophages treated with CTX (12.5 nM) or supernatants of CTX-treated macrophages and endothelial cell proliferation, migration and adhesion activities, and the capillary-like tube formation in the matrigel-3D matrix was measured. Angiogenic mediators (MMP-2, VEGF and TNF-α) were measured in the cell culture medium. Macrophages pre-treated with CTX and supernatant of CTX-treated macrophages inhibited EC proliferation, adhesion to its natural ligands, and migration (as evaluated in a wound-healing model and Time Lapse assay) activities. Decreased capillary-like tube formation and MMP-2, VEGF and TNF-α levels in the supernatant of macrophages treated with CTX was also described. CTX promotes macrophage reprogramming towards an antiangiogenic phenotype.