With neuropathy compared to those without neuropathy In the STZ model treatment was protective in the in vitro model

We showed that the exposition of DRG neurons monoculture to hyperglycaemia did not affect LY2835219 abmole neurite outgrowth, which did not differ from control monocultures. Conversely, neurite outgrowth significant decreased when DRG neuron monocultures were exposed to the medium of SC cultured in hyperglycaemia. This effect was mediated by the marked increase of VEGF in the medium of hyperglycaemiaconditioned SC monoculture, as confirmed by the dose-dependent impairment of neurite outgrowth after exposition of DRG coculture to VEGF. Previous studies showed that hyperglycaemia directly stimulates the secretion of VEGF in retinal Muiller cells and proximal tubular cells. Moreover it has been observed that VEGF protein level increased in DRG neurons and sciatic nerve axons from chronic STZ diabetic rats. These findings strengthened the hypothesis of a key role of VEGF also in the pathogenesis of DN, like already demonstrated in diabetic retinopathy and nephropathy. We demonstrated that hyperglycaemia inversely modified FLT1 protein level in DRG neuron monocultures and SC. FLT-1 is a cell-surface receptor for VEGF and may function as negative regulator limiting the amount of free VEGF and preventing its binding to VEGF receptor-2, the best characterized receptor and known to mediate most VEGF cellular responses. We also found that sFLT-1 was decreased. This soluble receptor lacks one transmembrane domain and may function as a decoy for VEGF. We speculated that in hyperglycaemia VEGF overruled sFLT-1 whose scavenger activity could not limit VEGF increase and its toxic effects. We showed that bevacizumab, a recombinant humanized monoclonal IgG1 antibody that binds VEGF and inhibits its biologic activity preventing the interaction to its receptors, was protective both in vitro and in vivo models of DN. Indeed, it reduced the level of free VEGF in the medium of DRG co-cultures exposed to hyperglycaemia and protected from the impairment of neurite outgrowth in a dose-dependent fashion. This was associated with the normalization of FLT-1 signaling between neurons and SC. Exposition of hyperglycaemia-conditioned DRG neuron monocultures to bevacizumab normalized FLT-1 mRNA with no change at the protein level. Conversely, exposition of hyperglycaemia-conditioned SC monocultures to bevacizumab increased FLT1 protein and reduced FLT-1 mRNA. Finally, we demonstrated that bevacizumab both protected and reversed neuropathy in STZ rats, confirming the neuroprotective effects of our in vitro studies. Indeed, preventive and therapeutic protocols of bevacizumab administration counteracted in a dosedependent fashion the pathological changes in thermal and mechanical thresholds, and in NCV which are hallmarks of diabetic neuropathy. Modulation of the VEGF/FLT1 signalling axis in vivo have to be further investigate in order to attribute efficacy of bevacizumab to a specific mechanism. Few and contradictory data are available on the role of VEGF in DN. Some works showed neuroprotective effects of VEGF on sensory and motor neurons, whereas others provided convincing evidence of direct toxic effects on nerves which are reversed by bevacizumab. A recent study reported significantly higher levels of serum VEGF in diabetic patients.

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