VEGF-A165b Attenuates Diabetic Neuropathic Pain In Vivo And Blocks Increased TRPV1 Activity And Phosphorylation Associated With High Glucose Conditions In Vitro

Diabetic neuropathy (DN) is a serious condition that affects up to 50% of patients suffering from diabetes mellitus (DM). A common symptom of DN is hypersensitivity to both painful and non-painful stimuli, which presents as chronic, spontaneous, shooting, burning, and tingling painful sensations. A potential cause of diabetic pain is the sensitisation of ion channels expressed on nociceptors, such as transient receptor vanilloid 1 (TRPV1). The vascular endothelial growth factor-A (VEGF-A) isoform, VEGF-A165b has analgesic effects in rodent models of neuropathythrough actions on TRPV1, which is up-regulated in diabetic neuropathy. We hypothesized that diabetic neuropathic pain is attributable to sensitisation of TRPV1, and that it would be ameliorated by rhVEGF-A165b treatment. In vivo: Diabetes was induced in adult female Sprague Dawley rats with streptozotocin (50mg/kg, ip) and maintained for 3-7 weeks. STZ treated rats were treated bi-weekly with rhVEGF-A165b (20ng/g, ip, n=9) or saline vehicle (n=9). Standard nociceptive tests assessed thermal and mechanical hypersensitivity. In vitro: Dorsal root ganglion (DRG) neurons were isolated from naïve (male Wistar) rats and cultured for up to 3 days. DRG neurons from naïve rats were cultured in either basal (10mM) or high (50mM) glucose ± VEGF-A165b. Intracellular calcium changes in response to capsaicin (TRPV1 agonist, 1µM) were measured using a high-throughput fluo-4 based assay. 50b11s, an immortalized embryonic DRG sensory neuronal cell line, were used for TRPV1 and phospho-TRPV1 S800 (PKC mediated phosphorylation site) expression in basal glucose (36mM) and high glucose (66mM) ± 2.5nM rhVEGF165b co-treatment. 50b11s require higher glucose levels for maintenance in culture than primary DRG neurons. After 3 weeks STZ + saline treated rats developed significant mechanical and thermal hypersensitivity when compared to control (n=11), which was significantly reduced with VEGF-A165b (mechanical withdrawal threshold [grams]: Naïve: 15.1±3.966 vs. STZ + saline: 2.3±0.788 p<0.001 vs. STZ + rhVEGF-A165b 13.1±1.831 p<0.01, thermal latency [fold change]: Naïve 1.0±0.129 vs. STZ + saline 0.6±0.029 p<0.05 vs. STZ + rhVEGF-A165b 1.0±0.104 p<0.05, two way ANOVA w/ post hoc Tukey). In vitro, there were significantly greater responses to 1µM capsaicin in DRG neurons cultured in 50mM glucose conditions compared to 10mM glucose (50mM AUC: 75.95 ± 13.3 fold change(s); 10mM AUC: 41.95 ± 5.2 fold change(s), n=4/group).The greater TRPV1-evoked responses in 50mM glucose were significantly reduced with 2.5nM VEGF-A165b treatment (24.25 ± 7.5fold change(s), n=3, p<0.01, one way ANOVA w/ post hoc Bonferroni). Total TRPV1 expression in 50b11s was unaffected by glucose concentration (36mM glucose: 0.977 ± 0.044fold change(s), n=6; 66mM glucose: 0.916 ± 0.044 fold change(s), n=6), or VEGF-A165b treatment (1.002 ± 0.058fold change(s), n=3). Culture in 66mM glucose significantly enhanced TRPV1 phosphorylation at serine 800 compared to 36mM glucose (66mM: 1.231 ± 0.058fold change(s), n=3; 36mM 0.843 ± 0.082fold change(s), n=3) and this was prevented by 2.5nM VEGF-A165b treatment (0.382 ± 0.053 fold change(s), n=3, p<0.001, one way ANOVA w/ post hoc Bonferroni). VEGF-A165b has anti-nociceptive actions STZ-induced diabetic neuropathy, and also blocks enhanced functional TRPV1 activity in isolated primary sensory neurons cultured in high glucose conditions. Enhanced TRPV1 function may be attributable to TRPV1 phosphorylation at the PKC-dependent serine 800-phosphorylation site, as phosphorylation is increased under similar culture conditions in immortalised sensory neurons. Interestingly VEGF-A165b treatment also blocks TRPV1 s800 phosphorylation in these cells. These findings suggest that PKC-dependent TRPV1 phosphorylation may contribute towards diabetic neuropathic pain and that the anti-nociceptive actions of VEGF-A165b may function by preventing this.