Effects of adipose derived stem cells in counteracting oxaliplatin-induced neuropathy: role of VEGF-A as possible applications

Oxaliplatin therapy of colorectal cancer induces a dose-dependent neuropathic syndrome in 50% of patients. Pharmacological treatments may offer limited relief; scientific efforts are being solicited to define a new therapeutic approach. Interestingly, adult mesenchymal stem cells offer a totipotent cellular source for replacing injured neural cells and at the same time represent a source of neuroprotective and anti-inflammatory mediators, opposing the effect of nerve damage. In our hands, adipose-derived stem cells (ASCs) injection (2x106 ASCs/rat i.v.) were able to counteract mechanical hyperalgesia induced by repeated oxaliplatin administration in rats (2.4 mg kg-1 i.p. for a total of 10 administrations). This effect reached a maximum 6h after ASCs administration and lasted up to 72h. Subsequent ASCs administrations induced a reduction of hypersensitivity, with a similar efficacy trend over time. Investigating a possible mechanism of action by which ASCs exert their effect, 2x106 ASCs labeled with 1 µM of the fluorescent probe 5-(and-6-9-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine were injected in order to evaluate the localization of ASCs in the rat body. Labelled ASCs were detectable in the bloodstream 1 and 3 h after injection, the percentage gradually decreased, 24 h after administration no cells were found. At this time, ASCs were detected in the liver digested homogenate. No ASCs were found in the central nervous system and in lungs. VEGF-A, EGF and TGF-β were assayed in plasma. EGF and TGF-β were not altered by oxaliplatin or ASCs treatments. On the contrary, VEGF-A concentration significantly increased in oxaliplatin-treated rats in comparison to the control group whereas ASCs were able to counteract this alteration, suggesting both a possible implication of VEGF modulation in the development of neuropathic pain and in ASCs pain relieving mechanism. This hypothesis was strengthened by the reduction of oxaliplatin-induced hyperalgesia after an acute i.p. administration of the VEGF-antibody bevacizumab (dose-dependently, 1-15 mg kg-1). Moreover, plantar injection of the pain-related isoform VEGF165b (10-100 ng) in naïve rats, significantly decreased the pain threshold up to 3 h after administration in a bevacizumab-reverted manner (15 mg kg-1). VEGF165b (30-100 ng) dose-dependently, significantly reduced the weight tolerated on the posterior paw also after an intrathecal administration. These data led us to further investigate the role of VEGF-A in modulating pain signaling and the correlation between ASCs anti-nociceptive efficacy and VEGF-A modulation in central nervous system. As evaluated by western blot analysis, oxaliplatin repeated treatment significantly increased VEGF165b expression in spinal cord and PAG while in DRG, cortex and thalamus the protein levels were not influenced with respect to control group. ASCs i.v. injected significantly counteracted the oxaliplatin-dependent VEGF165b increase only in spinal cord. Accordingly, an intrathecal administration of ASCs (75x103/rat) was able to revert hyperalgesia induced by repeated oxaliplatin treatments starting from 1 h after the injection and up to 6 h. To further investigate the link between central VEGF-A and the modulation of pain perception, the selective neutralization of VEGF-A and VEGF165b was performed both in oxaliplatin-treated and in control rats. The specific antibody against VEGF165b, i.t. injected (7.5 μg/rat), significantly decrease oxaliplatin-induced hyperalgesia with similar efficacy to bevacizumab (45 μg/rat i.t. injected) but with higher potency. The modulation of VEGF-A is suggested as a key mechanism in the complex response orchestrated by stem cells against neuropathy and the role of ASCs “secretome” as effector of their efficacy is highlighted. For this reason, an artificial and biocompatible niche in which ASCs can be maintained was set up and the efficacy of this engineered niche in relieving oxaliplatin-induced neuropathic pain was tested. As physiologically occurs in tissues, the artificial niches should promote ASCs proliferation, ensuring the maintenance of their stemness and meanwhile permit a bidirectional flux of solutes from the inner and outer environment without a direct delivery of ASCs. For this purpose, 4x106 rASCs were encapsulated into each artificial niche (AlgiMatrix® sponge), the day after AlgiMatrix® sponges were implanted s.c. in the back of neuropathic rats (two sponges per rat). Encapsulated rASCs significantly reduced mechanical hypersensitivity induced by oxaliplatin treatment beginning 6h after the surgery. The effect on hypersensitivity remained constant for 7 days after AlgiMatrix® sponges implantation and disappeared at 8th day. Enclose ASCs in an artificial niche allows to exploit their modulatory capabilities in response to tissue injuries reducing possible side effects associated to stem cell differentiation. So, the present data suggest an alternative approach in the use of ASCs for the treatment of oxaliplatin-induced neuropathic pain.