Furthermore, inhibition of either Rac1 or NADPH oxidase activity protected from vascular injury in this system. Our data strongly suggest that most of the cellular effects of HG are due to a modest increase in Rac1 activation, which is in agreement with other studies that have shown that moderate Rac1 activation is sufficient to decrease migration speed and directionality, as well as to promote an increase in the number of protrusions per cell. However, the increased Rac1 activity may lead to its mislocalization, which would amply the effect. Our data also shows that HG increases RhoA activity. Rac1 and RhoA are reciprocally regulated by a negative feedback loop, which may implicate NAPDH oxidase. However, the increase in RhoA activity is not XAV939 specifically dependent on the glucose metabolism, since the osmotic control also displayed similar increase. Osmotic pressure applies mechanical stress to the cellular cortex, which may contribute to this activation similar to mechanical tension which, applied to the cell membrane, increases RhoA activation, bypassing the requirement of low Rac activity and thus breaking down the Rac-RhoA feedback loop. However, RhoA is also associated to the deleterious effects of diabetes, particularly in mesangial cells, as well as in several organs that are targeted by diabetes. The crosstalk between Rho GTPases and other pathways under HG conditions cannot be ruled out. For example, the inhibition of the nutrient-sensing mammalian target of rapamycin pathway inhibits RhoA and Rac1 activity, affecting cell motility. This pathway is activated by glucose and, in several cell types, its activation is required for acquiring a senescent phenotype. One of the phenotypical alterations promoted by HG in fibroblasts was an increased cell size, which is also observed in senescent cells, due to hypertrophy. Interestingly, there is a positive feedback between the TOR pathway and ROS generation. ROS are generated in mitochondria in response to glucose, stimulating the mTOR pathway. TOR signaling, on the other hand, may regulate mitochondrial proteome dynamics; it was shown that reduced TOR signaling increases mitochondrial oxygen consumption and decreases ROS generation. Thus, the effect of NAC reversing the HG phenotype might be at least partially related to the inhibition of the mTOR pathway. In summary, this study provides novel mechanistic insight into the effects of high glucose on cell migration, which is a likely contributor to the defects in wound healing often observed in diabetic patients.