This manuscript presents a body of evidence implicating VEGF signaling in the enhanced innervation of the urinary bladders following instillation of BCG in mice. Qualitatively these results add to a list of evidence implicating the VEGF pathway in the bladder responses to BCG. A quantitative analysis of inflammatory infiltrate and vascular plasticity indicates possible alternative pathways activated by BCG and VEGF.
The nervous and vascular systems share several anatomical parallels. Both systems utilize a complex branching network of neuronal cells or blood vessels reaching all regions of the body. The anatomical similarity of the nervous and vascular systems suggests that axons might guide blood vessels and vice versa . Indeed, signal molecules produced by peripheral neuronal cells, such as VEGF , guide blood vessels , and signals from vessels, such as the neurotrophins NGF and NT-3, are required for, and orchestrate extension of neurons adjacent to vessels . In this manner, the neuronal and vascular systems are well organized and coordinated in normal adult tissues. However, in chronic inflammatory states particularly in the LUT, little is known about how the nerve-vessel relationship functions and whether it could underlie the chronic pain syndrome observed in certain patients.
Interest in guidance molecules, and particularly VEGF, modulating both vascular and neuronal pathology is emerging. VEGF levels are associated with alterations in the bladder vascular system. VEGF is increased in bladders of patients with painful bladder syndrome, and it is associated with glomerulations on hydrodistension . However, increased bladder VEGF is not observed in patients who failed to show petechial bleeding or in controls . In addition, VEGFR-1 and NRP2 expressions are reduced in bladder biopsies from patients with cystitis .
We reasoned that pro-inflammatory stimuli known to increase VEGF, and in particular NRP expression, might also modulate inflammation-induced nerve plasticity. The contribution of chronic inflammation to peripheral nerve plasticity was investigated in a murine model using BCG instillations that it is known to increase signaling molecules, such as IL-17  and VEGF , that are necessary for inducing neuronal plasticity. Our results confirmed our hypothesis that intravesical instillation of BCG increased the density of PGP9.5-, SP-, TRPV1-, and CGRP-IR within the bladder wall. For some parameters, the numbers of sensory nerves identified closely parallels numbers of PGP9.5 immunoreactive nerves, which (accepting the limitations of sensitivity of staining techniques) could indicate that most PGP9.5 fibers observed were peptidergic sensory nerves. However, further studies on sympathetic nerve staining and non-peptidergic nerve afferents are necessary to determine the whole extend of BCG- and VEGF-induced neuronal plasticity.
A limitation of our experimental conditions is that concentrations of neutralizing antibodies and the pro-inflammatory stimuli were probably different among the different layers of the urinary bladder. The antibodies were systemically administered, whereas pro-inflammatory stimuli were instilled into the bladder. Therefore, we analyzed the effects on both the sub-urothelium and detrusor muscle, and the results obtained seem to reflect differences in tissues that are in closer proximity to the stimulus. Interestingly, BCG induced an increase of TRPV1-IR in the sub-urothelium but not in the detrusor smooth muscle, whereas PGP9.5-IR was found increased in both layers. As BCG favors an increase of inflammatory cells primarily in the sub-urothelial layer when compared to the detrusor muscle, these results suggest TRPV1-IR, but not PGP9.5-IR, is associated with the degree of inflammatory infiltrate. In this context, in the absence of inflammatory cell infiltrate, such as in IL-17 knockout mice, a significant decrease in mechanical pain hypersensitivity was observed . In contrast, when neutrophil infiltration was increased by administration of IL-17, a concomitant increase in neuropathic pain was observed . Furthermore, depletion of circulating neutrophils at the time of nerve injury significantly attenuated the induction of hyperalgesia .
Besides neutrophils and macrophages that were quantified in the present study, other inflammatory cells should be considered to have an action in neural plasticity. In particular, NRPs are recognized as a new marker for regulatory T (Treg) cells [45, 46] and are expressed in antigen presenting cells and effector cells [47, 48]. In addition to NRPs, SEMAs seem to participate in inflammation as it has been suggested that macrophages and fibroblasts secrete SEMAs that may be responsible for reduced sympathetic innervation . Therefore, it is fair to propose that one possible mechanism by which chronic BCG instillation induces bladder neuronal plasticity is by attracting inflammatory cells that will contribute to increased tissue levels of VEGF or SEMA.
At this time, there is not definitive evidence correlating the type of inflammatory cell involved in the regulation of bladder sensory nerve plasticity. However, given the known trophic effects of VEGF on neurite growth [34, 50] prolonged survival of neurons [51, 52], and re-enervation following local nerve damage [53, 54], it is fair to propose that inflammatory cells producing VEGF may be involved in the observed neural plasticity. This new appreciation of VEGF signaling in bladder inflammation is supported by emerging evidence that levels of various VEGF subtypes are, in general, increased at the site of inflammation, and that infiltrating lymphocytes and other inflammatory cells represent an additional source of VEGF .
The present work introduces intriguing results regarding NRPs. As NRP1 has a domain structure strikingly similar to that of NRP2 , we first tried to disrupt binding of VEGF and SEMA to NRP1 using blocking antibodies that were engineered to block either the a1-a2 domain or b1-b2 domains of NRP1. Both antibodies, although proven to reduce BCG-induced inflammation, angiogenesis, and vascular remodeling , failed to alter the effects of BCG on nerve density. This suggests that either the antibodies are specific for impairing inflammation-induced angiogenesis or that NRP1 may not participate in mechanisms underlying neuronal plasticity in the urinary bladder. An alternative explanation is based on findings that spatial gradients of Sema3A and VEGF may promote differential NRP1 binding . Indeed, vessels expressing high levels of Sema3A favor NRP1-PlexinA1 signaling, producing chemorepulsive cues limiting sympathetic neurite outgrowth and vascular enervation, while low Sema3A expressing vessels favor NRP1-VEGFR2 signaling that provides chemoattractive cues for sympathetic neurite outgrowth and vascular enervation .
The unexpected increase in nerve density after NRP2B administration deserves further investigation and studies are underway in our laboratory to define the mechanism/s involved in bladder responses to this antibody. It is also interesting that NRP2B alone did not induce alteration of nerve density. However, when given concomitantly with BCG, NRP2B induced an overwhelming increase in bladder nerve fibers. One possible explanation is that NRP2B might potentiate nerve plasticity by activating angiogenesis or increasing the migration of inflammatory cells such as neutrophils and macrophages. Our preliminary results indicate that NRP2B alone does not alter bladder blood vessel density or the number of inflammatory cells (data not shown). However, NRP2B does increase both F4/80+ macrophages and MPO+ neutrophils in response to BCG (data not shown).
It seems that in addition to activate the VEGF signaling pathway, BCG may have induced an increase in SEMAs. This would explain the finding that NRP2B further increased the number of nerves when administered concomitant to BCG. As SEMAs are known for strong chemorepulsion, these results suggest that blockade of NRP2 reduces SEMA participation and the consequent repulsion of neurons. Although NRP2B antibody was originally developed to target the coagulation V/VII factor (b1-b2) domains of NRP2 which are required for VEGF-C binding to NRPs , and the b1-b2 domains do not directly engage SEMA, this antibody could decrease SEMA binding by preventing NRP dimerization that is necessary for accommodating SEMA domains that pack tightly together at this interface .
It is possible that other secreted semaphorins binding to NRP2 alone are responsible for the chemorepulsion and the consequent effect of NRP2B antibody on neural plasticity. NRP2 is known to be required for mediating repulsive actions of Sema3B, 3C, and 3F, whereas NRP1 is known to be required for Sema3A function. In fact, Sema3B and Sema3F seem to require only NRP2, not NRP1, to elicit their effects, whereas Sema3C may require both NRP1 and NRP2 . Another piece of evidence indicating that NRP2 may play a role in altered nerve plasticity is the finding that peripheral nerve regeneration is delayed in NRP2-deficient mice , indicating that this guidance molecule facilitates peripheral-nerve axonal regeneration.
We also found evidence that target-derived VEGF165 or VEGF121 plays a previously unrecognized role in promoting growth of bladder nerves and inflammation. A role for VEGF in inflammation has been postulated. However, instillation of VEGF into the mouse bladder represents direct evidence that VEGF induces inflammation and that this new animal model can be used to investigate the effects of elevated levels of VEGF on bladder neuronal and vascular plasticity.
A fundamental question raised by the present findings is whether the increased innervation occurred as a consequence of angiogenesis or inflammation. It is known that innervation typically accompanies blood vessels  which made it important to determine whether the observed effects on nerves are direct, or mediated through effects on angiogenesis. This was a difficult topic to address facing the evidence indicating an overlap between inflammation and angiogenesis. We found that increased microvessel density (MVD) was part of the bladder responses to chronic instillations of BCG, VEGF165, and VEGF121. However, BCG did not induce a significant endothelial cell proliferations as indicated by KI67 and therefore, the increased MVD observed in response to BCG was probably related to the intense vasodilation known to be part of the inflammatory response. In contrast, proliferating endothelial cells were only observed in VEGF-treated tissues and, therefore, it seems that angiogenesis underlies the bladder responses to VEGF, as indicated by an increased KI67 expression on CD31+ endothelial cells .
Although an increase in nerve density, particularly those expressing TRPV1-IR, has been proposed to underline pain sensation and neurogenic detrusor overactivity based on findings that desensitization of afferents with capsaicin and resiniferatoxin decrease pain and detrusor instability , the present work did not explore whether the increased nerve density corresponded to an increased function of the sensory system. The primary goal of the present work was to explore putative mechanisms involved in inflammation-induced neural plasticity before conducting a detailed study on nerve function. Our results were focused on the target organ, and future studies should include the consequences of VEGF instillation on more central neurons such as those of the dorsal root ganglia. In this context, it should be recognized that while plasticity of the central nervous system (in response to stimuli) and regeneration (in response to injury) are mainly based on adaptive changes in neural circuits and synaptic reorganization, plasticity of the peripheral nervous system is predominantly based on axonal re-growth and neuron addition .