Advanced glycation endproducts produced by in vitro glycation of type I collagen modulate the functional and secretory behavior of dorsal root ganglion cells cultivated in two-dimensional system.

Advanced glycation end-products (AGEs) are proteins/lipids that are glycated upon sugar exposure and are often increased during inflammatory diseases such as osteoarthritis and neurodegenerative disorders. Here, we developed an extracellular matrix (ECM) using glycated type I collagen (ECM-GC), which produced similar levels of AGEs to those detected in the sera of arthritic mice. In order to determine whether AGEs were sufficient to stimulate sensory neurons, dorsal root ganglia (DRGs) cells were cultured on ECM-GC or ECM-NC-coated plates. ECM-GC or ECM-NC were favorable for DRG cells expansion. However, ECM-GC cultivated neurons displayed thinner F-actin filaments, rounded morphology, and reduced neuron interconnection compared to ECM-NC. In addition, ECM-GC did not affect RAGE expression levels in the neurons, although induced rapid p38, MAPK and ERK activation. Finally, ECM-GC stimulated the secretion of nitrite and TNF-α by DRG cells. Taken together, our in vitro glycated ECM model suitably mimics the in vivo microenvironment of inflammatory disorders and provides new insights into the role of ECM impairment as a nociceptive stimulus.

Chondroitin Sulfate Impairs Neural Stem Cell Migration Through ROCK Activation.

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.

High glucose-mediated oxidative stress impairs cell migration.

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.