Sensory neuronal sensitisation occurs through HMGB-1-RAGE and TRPV1 in high-glucose conditions.

Many potential causes for painful diabetic neuropathy have been proposed including actions of cytokines and growth factors. High mobility group protein B1 (HMGB1) is a RAGE (also known as AGER) agonist whose levels are increased in diabetes and that contributes to pain by modulating peripheral inflammatory responses. HMGB1 enhances nociceptive behaviour in naïve animals through an unknown mechanism. We tested the hypothesis that HMGB1 causes pain through direct neuronal activation of RAGE and alteration of nociceptive neuronal responsiveness. HMGB1 and RAGE expression were increased in skin and primary sensory (dorsal root ganglion, DRG) neurons of diabetic rats at times when pain behaviour was enhanced. Agonist-evoked TRPV1-mediated Ca2+ responses increased in cultured DRG neurons from diabetic rats and in neurons from naïve rats exposed to high glucose concentrations. HMGB1-mediated increases in TRPV1-evoked Ca2+ responses in DRG neurons were RAGE- and PKC-dependent, and this was blocked by co-administration of the growth factor splice variant VEGF-A165b. Pain behaviour and the DRG RAGE expression increases were blocked by VEGF-A165b treatment of diabetic rats in vivo Hence, we conclude that HMGB1-RAGE activation sensitises DRG neurons in vitro, and that VEGF-A165b blocks HMGB-1-RAGE DRG activation, which may contribute to its analgesic properties in vivo.

Alternative RNA splicing: contribution to pain and potential therapeutic strategy.

Since the sequencing of metazoan genomes began, it has become clear that the number of expressed proteins far exceeds the number of genes. It is now estimated that more than 98% of human genes give rise to multiple proteins through alternative pre-mRNA splicing. In this review, we highlight the known alternative splice variants of many channels, receptors, and growth factors that are important in nociception and pain. Recently, pharmacological control of alternative splicing has been proposed as potential therapy in cancer, wet age-related macular degeneration, retroviral infections, and pain. Thus, we also consider the effects that known splice variants of molecules key to nociception/pain have on nociceptive processing and/or analgesic action, and the potential for control of alternative pre-mRNA splicing as a novel analgesic strategy.

Vascular endothelial growth factor-A165b prevents diabetic neuropathic pain and sensory neuronal degeneration.

Diabetic peripheral neuropathy affects up to half of diabetic patients. This neuronal damage leads to sensory disturbances, including allodynia and hyperalgesia. Many growth factors have been suggested as useful treatments for prevention of neurodegeneration, including the vascular endothelial growth factor (VEGF) family. VEGF-A is generated as two alternative splice variant families. The most widely studied isoform, VEGF-A165a is both pro-angiogenic and neuroprotective, but pro-nociceptive and increases vascular permeability in animal models. Streptozotocin (STZ)-induced diabetic rats develop both hyperglycaemia and many of the resulting diabetic complications seen in patients, including peripheral neuropathy. In the present study, we show that the anti-angiogenic VEGF-A splice variant, VEGF-A165b, is also a potential therapeutic for diabetic neuropathy. Seven weeks of VEGF-A165b treatment in diabetic rats reversed enhanced pain behaviour in multiple behavioural paradigms and was neuroprotective, reducing hyperglycaemia-induced activated caspase 3 (AC3) levels in sensory neuronal subsets, epidermal sensory nerve fibre loss and aberrant sciatic nerve morphology. Furthermore, VEGF-A165b inhibited a STZ-induced increase in Evans Blue extravasation in dorsal root ganglia (DRG), saphenous nerve and plantar skin of the hind paw. Increased transient receptor potential ankyrin 1 (TRPA1) channel activity is associated with the onset of diabetic neuropathy. VEGF-A165b also prevented hyperglycaemia-enhanced TRPA1 activity in an in vitro sensory neuronal cell line indicating a novel direct neuronal mechanism that could underlie the anti-nociceptive effect observed in vivo. These results demonstrate that in a model of Type I diabetes VEGF-A165b attenuates altered pain behaviour and prevents neuronal stress, possibly through an effect on TRPA1 activity.

Novel mechanisms of resistance to vemurafenib in melanoma - V600E B-Raf reversion and switching VEGF-A splice isoform expression.

Targeting activating mutations in the proto-oncogene B-Raf, in melanoma, has led to increases in progression free survival. Treatment with vemurafenib, which inhibits the most common activating-mutated form of B-Raf (B-Raf(V600E)), eventually results in resistance to therapy. VEGF-A is the principal driver of angiogenesis in primary and metastatic lesions. The bioactivity of VEGF-A is dependent upon alternative RNA splicing and pro-angiogenic isoforms of VEGF-A are upregulated in many disease states dependent upon angiogenesis, including cancers. Using techniques including RT-PCR, Western blotting, ELISA and luciferase reporter assays, the effect of vemurafenib on proliferation, ERK1/2 phosphorylation and the levels of pro- and anti-angiogenic VEGF-A isoforms was investigated in melanoma cell types expressing either wild-type B-Raf or B-Raf(V600E), including a primary melanoma culture derived from a highly vascularised and active nodule taken from a patient with a V600E mutant melanoma. The primary melanoma culture was characterised and found to have reverted to wild-type B-Raf. In B-Raf(V600E) A375 cells ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA, total VEGF-A protein expression and VEGF-A 3'UTR activity were all decreased in a concentration-dependent manner by vemurafenib. Conversely vemurafenib treatment of wild-type B-Raf cells significantly increased ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA and total VEGF-A expression in a concentration-dependent manner. A switch to pro-angiogenic VEGF-A isoforms, with a concomitant upregulation of expression by increasing VEGF-A mRNA stability, may be an additional oncogenic and pathological mechanism in B-Raf(V600E) melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-Raf(V600E) to wild-type in an active melanoma nodule taken from a V600E-positive patient and continued vemurafenib treatment for this patient is likely to have had a detrimental effect by promoting B-Raf(WT) activity.

VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro.

Vascular endothelial growth factor (VEGF) A is generated as two isoform families by alternative RNA splicing, represented by VEGF-A165a and VEGF-A165b. These isoforms have opposing actions on vascular permeability, angiogenesis, and vasodilatation. The proangiogenic VEGF-A165a isoform is neuroprotective in hippocampal, dorsal root ganglia, and retinal neurons, but its propermeability, vasodilatatory, and angiogenic properties limit its therapeutic usefulness. In contrast, a neuroprotective effect of endogenous VEGF-A165b on neurons would be advantageous for neurodegenerative pathologies. Endogenous expression of human and rat VEGF-A165b was detected in hippocampal and cortical neurons. VEGF-A165b formed a significant proportion of total VEGF-A in rat brain. Recombinant human VEGF-A165b exerted neuroprotective effects in response to multiple insults, including glutamatergic excitotoxicity in hippocampal neurons, chemotherapy-induced cytotoxicity of dorsal root ganglion neurons, and retinal ganglion cells (RGCs) in rat retinal ischemia-reperfusion injury in vivo. Neuroprotection was dependent on VEGFR2 and MEK1/2 activation but not on p38 or phosphatidylinositol 3-kinase activation. Recombinant human VEGF-A165b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro through VEGFR2, MEK1/2, and inhibition of caspase-3 induction. VEGF-A165b may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral neuropathy. The endogenous nature of VEGF-A165b expression suggests that non-isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons.

VEGF-A165b is cytoprotective and antiangiogenic in the retina.

PURPOSE: A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A(165), the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A(165)b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF(165)b and its effect on retinal epithelial and endothelial cell survival. METHODS: VEGF-A(165)b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A(165)b. RESULTS: VEGF-A(165)b dose dependently inhibited angiogenesis (IC(50), 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A(165) across monolayers in culture (IC(50), 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A(165)b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC(50), 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A(165)b. CONCLUSIONS: The survival effects of VEGF-A(165)b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A(165)b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.