VEGF-A/VEGFRs system in neuropathies: a crossroad between pain and neuroprotection

Vascular Endothelial Growth Factor (VEGF) is a family of signal proteins produced by different cells, that stimulates the formation of blood vessels. VEGF-A is the most studied member of its family. In addition to its well-known pro-angiogenic properties, it also directly influences neuronal and glial biological processes, exerting trophic and signaling functions in nervous tissue. In recent years, the involvement of the VEGF family in pain signaling is emerging, highlighting the opportunity of a new possible pharmacological target and making urgent the knowledge of its role in the pathophysiological mechanisms of algic sensitivity. For these reasons, the purpose of this thesis was to investigate the involvement of VEGF-A and its receptors VEGFR-1 and VEGFR-2 in pain perception, and the possible role of this growth factor in neuroprotection. In naïve mice, intrathecal infusion of VEGF165b (3- 10 and 30 ng/5 µL- a most representative member of VEGF-A) induced dose-dependent noxious hypersensitivity, assessed by pain threshold measurement, mediated by its VEGFR-1. The involvement of VEGFR-1 was confirmed by both using selective ligands (PlGF and VEGF-E that bind VEGFR-1 and VEGFR-2 respectively) and receptor blockers (mAb D16F7 specific for VEGFR-1 and DC101 for VEGFR-2), and from the silencing of the two VEGFRs by siRNAs in the lumbar spinal cord. In addition, to deepen the molecular mechanism underlying the painful action of VEGF-A, the immunofluorescence analysis showed that VEGFR-1 is more expressed on neuronal rather than astrocytic cells. Consequentially, in the electrophysiological study, VEGF165b stimulated the activity of spinal nociceptive neurons via VEGFR-1. Furthermore, in the dorsal horn of the spinal cord, immunofluorescence analysis revealed that VEGF-A increased in astrocytes from animals with oxaliplatin-induced neuropathy, compared to microglia and neurons, suggesting that this cell population is the source of the effective pain mediator. In addition, confocal microscopy confirmed the expression of VEGF-A in astrocytes, separately from its vascular component. To investigate the relevance of this result, we selectively silenced astrocytic VEGF-A via shRNAmir in spinal cord of neuropathic animals, resulting in a block of the development of chemotherapy-induced neuropathic pain. In addition, anti-VEGFR-1 mAb D16F7 effectively relieved neuropathic pain induced by various chemotherapeutic agents. Following these data, to further investigate the mechanisms of pain modulation and to study the neuroprotective component of VEGF-A in nervous tissue, we used the organotypic spinal cord slice. After fourteen days of cultivation, the slices were analyzed by immunofluorescence analysis with GFAP and NeuN markers to confirm the maintenance of cell morphology and structural organization of the spinal cord; in addition, using RECA-1 as endothelial marker, we highlighted a significant reduction of the normal vascular network. At this point, we focused our attention on three "key" factors that play important role in the development and maintenance of pain: Calcitonin gene-related peptide (CGRP), widely distributed in peripheral and central nervous system and its receptors are expressed in pain pathways; Substance P, involved in the onset and modulation of different types of pain; Glutamate, that showed a pivotal role in pain sensation and transmission. Treatment with both oxaliplatin (10 µM) and VEGF65b (100 ng/mL) enhanced the release of CGRP and Substance P in the culture medium of the slices compared to control; co-treatment with D16F7 (300 ng/mL), but not with DC101 (10 ng/mL), prevented the release of both two pro-algic factors. Measuring the mRNA of EAAT1 and EAAT2, the reduction in gene expression of the two glutamate transporters caused by VEGF165b, as well as by oxaliplatin, was improved by VEGFR-1 blocker. From the toxicity studies, we observed that oxaliplatin causes a dose-dependent neurotoxicity and alteration of neurons morphology expressed as a reduction in fluorescence intensity and in the number of NeuN+ cells, after 24 hours incubation. Moreover, activation of astrocytes (evaluated by immunofluorescence staining) was observed. The co-treatment with VEGF165b showed neuroprotection of the nervous tissue assessed by PI fluorescence, reduction of astrogliosis and neuronal alterations caused by oxaliplatin. To investigate the molecular mechanism underlying this neuroprotective effect, we analyzed the role of VEGFR-1 and VEGFR-2 by using PlGF and VEGF-E as their specific ligand and also D16F7 and DC101 as receptor blockers. Quantitative analysis of PI fluorescence showed that VEGFR-2 is involved in VEGF-A-mediated neuroprotection. Ultimately, since the existence of astrocytic VEGF-A is reported in the literature, as confirmed by our in vivo results, we treated the slices with fluorocitrate, a glial metabolism blocker, to evaluate its effect in physiological and pathological conditions. Our results showed that fluoricitrate (80 µM) was able to reduce both VEGF-A baseline release and that induced by oxaliplatin treatment. Moreover, the astrocytic inhibition caused an increase in PI fluorescence at all times considered, as expected, worsening the toxicity due to oxaliplatin after three hours of treatment. The addition of exogenous VEGF165b reduces the toxicity caused by oxaliplatin and fluorocitrate after 24 hours of treatment. In conclusion, this thesis highlighted that VEGF-A released by astrocytes is a new actor in the complex neuron-glia network that oversees physiological and pathological pain, and mAb D16F7 exerts a potent painkiller action in different models of chemotherapy-induced neuropathic pain. Furthermore, the use of organotypic slices of the spinal cord has allowed to deepen the dichotomy between the proalgic and neuroprotective action of VEGF-A, highlighting that VEGFR-1 could be a promising therapeutic target in the modulation of chemotherapy-induced neuropathic pain, without blocking the protective component of the growth factor.