Fig. 6. Expression of VEGF-A165a and VEGF-A165b in rat DRG. A. VEGF-A165b represents ~70% of total VEGF-A expression in DRG. B. In one human DRG VEGF-A165b represented a similar proportion of total VEGF-A expression to that seen in the rat. C. VEGF-A165b is expressed in neurons in embryonic human spinal cord and DRG. Higher magnification images are derived from the boxes in the top image and are left: DRG and right: spinal cord ventral horn. D. VEGF-A165b is expressed in a proportion of rat DRG neurons, with overlap with the nociceptive markers TrkA and a small colocalization with IB4. Scale bar = 75 m. High power images of a single neuron showing colocalization of VEGF-A165b and TrkA. Scale bar = 50 m. mRNA splicing and maturation. Of the mammalian target RNAs affected by SRPK1/2 and SRSF1-controlled splicing, none have been previously implicated in pain or PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19841886 nociception, other than VEGF-A. As pre-mRNA splicing inhibition affected the balance of endogenous VEGF-A isoforms and nociception, and exogenous VEGF-A isoforms modulated behaviors and neuronal properties in a similar fashion, we hypothesize that it is the balance of VEGF-Axxxa and VEGF-Axxxb that determines the net effect on nociception. A slight disruption in this balance can have profound effects on VEGFR2 function as both receptor number and intracellular signaling mechanisms are altered. VEGF-A165a and VEGF-A165b have the same binding affinities 
to VEGFR2. However, when the two isoforms are equimolar or VEGFA165b is in excess ), VEGF-A165b can reduce VEGF-A165a actions by ~ 95%. This is brought about by competitive antagonism at VEGFR2, and reduction in receptor number. This complex mechanism can explain why local alteration of alternative RNA splicing, with a N 60% reduction in VEGFA165a mRNA in skin, induced hypoalgesia in normal animals whereas systemic low concentration VEGF-A165b had little effect. Conversely, increasing VEGF-A165a using systemic exogenous recombinant protein had clear pro-nociceptive effects on both behavior and neurons. Increasing local VEGF-A165a had a robust action on a sub-population of small unmyelinated somatic nociceptors that express functional VEGF receptors and TRPV1 receptors, increasing spontaneous firing and mechanically-evoked activity, and lowering activation thresholds, all changes indicative of peripheral sensitization of sensory neurons. All of these changes, particularly increased spontaneous firing, increase afferent barrage and induce central sensitization in the spinal cord and higher centers, leading to altered pain behaviors . Peripheral administration of VEGF-A165a had rapid effects on primary afferents in vivo, suggestive of direct VEGF-A effects on neurons. This is supported by our data that show increased R.P. Hulse et al. / Neurobiology of Disease 71 245259 255 Fig. 7. VEGF-A isoforms alter nociception in a TRPv1 dependent manner. A. Systemic TRPV1 antagonism with SB366791 in mice resulted in inhibition of rhVEGF-A165a-induced mechanical allodynia. Arrows denote time of drug administration. B. TRPV1 knockout mice did not develop rhVEGF-A165a-induced mechanical allodynia, in contrast to wild-type strain Birinapant biological activity matched controls. C. TRPV1 was co-expressed with VEGFR2 in sensory dorsal root ganglia sensory neurons. D. Local administration of VEGF165a + vehicle into the plantar hindpaw resulted in a reduction in mechanical withdrawal values, which was blocked by co-administration of the TRPV1 antagonist SB366791. Y1175 phosphorylation o