Figures ?Figures6A6A and ?and6B6B are low magnification micrographs in order to illustrate the distribution of TRPV1-positive fibers in several layers of the urinary bladder isolated from control (PBS) and inflamed bladders (BCG), respectively. (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. Results The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF VU661013 neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density. NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed VU661013 to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. Conclusions For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon. Background It is highly likely that sensory dysfunction is involved in various disorders of the lower urinary tract (LUT) including neurogenic bladder, outflow obstruction, idiopathic detrusor instability, overactive bladder, painful bladder syndrome, and diabetic neuropathy involving the bladder. In addition, chronic pathological conditions that cause tissue irritation or inflammation can alter the properties of sensory pathways, leading to a reduction in pain threshold and/or an amplification of painful sensation (hyperalgesia) [1]. Depending on the pathology, several mediators and their respective receptors have been proposed to modulate peripheral nerve plasticity in the LUT, including but not limited to: purinergic receptors in general [2] or P2X receptor in particular [3], transient receptor Adam30 potential vanilloid subfamily 1 VU661013 (TRPV1) [1], substance P acting on NK1 receptors [4], protease activated receptors [5], and nerve growth factor and its receptors [6]. The new hypothesis being tested in this manuscript is that increased levels of VEGF observed during bladder inflammation provoke nerve plasticity. This hypothesis is based on evidence indicating that nerves and blood vessels are associated, follow a common molecular pathway during development, and key molecules responsible for their development may continue to control their plasticity in adulthood [7]. The finding that mutant mice (neurogenin1/neurogenin2 double knockout embryos) lacking sensory nerves also present disorganized blood vessel branching [8], suggests that local signals such as VEGF supplied by nerve fibers, may provide a cue that determines blood vessel patterning. In contrast, administration of VEGF can support and enhance the growth of regenerating nerve fibers, probably through a combination of angiogenic, neurotrophic, and neuroprotective effects [9]. In this context, many proteins that were originally discovered to be required for axon guidance have recently been implicated in the development of the vascular [10] and lymphatic systems [11]. Perhaps the most striking observation is that angiogenic factors, when deregulated, contribute to various neurological disorders, such as neurodegeneration. The prototypic example of this cross-talk between nerves and vessels is the vascular endothelial growth factor, VEGF [12]. Although originally described as a key angiogenic factor, it is now well established that VEGF also plays a crucial role in development of the nervous system [12]. Among the neuronal guidance molecules, neuropilins (NRPs) and plexins, and their ligands, semaphorins and VEGF have been extensively studied in the central nervous system. They represent large families of molecules that can transduce signals essential for the regulation of neuronal repulsion and attraction, cell shape, motility, and cell-cell interactions [13-15]. Plexins are similar to the Toll-like receptors (TLRs) in their evolutionary conservation from flies to mammals. In particular, plexin A4 has been shown to be required for bacteria and LPS to engage TLR and trigger the downstream signal transduction pathway including activation of Rac1, c-Jun N-terminal kinase, NF-kB and AP-1 [16]. In addition, plexin-A4.
