The major findings of the present manuscript are: 1) the instillation of VEGF produces an increase in sensory nerve density, presumably by nerve sprouting, that reaches a maximum at 2 weeks and declines by 4 weeks; 2) in addition to significant increase in sensory nerve fibers, VEGF increases the density of bladder cholinergic nerve fibers; and 3) the increase in nerve density produced by VEGF results in altered bladder function and visceral sensitivity. The unique feature of our findings is that VEGF produces an increase in nerve density via urothelium.
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 [
22]. Indeed, signal molecules produced by peripheral neuronal cells, such as VEGF [
12], guide blood vessels [
14] and signals from vessels, such as the neurotrophins NGF and NT-3, are required for, and orchestrate extension of neurons adjacent to vessels [
23]. 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 patients with disorders of the lower urinary tract. In this context, this manuscript presents a body of evidence implicating VEGF signaling in the enhanced innervation of the urinary bladders in mice and the consequent alteration in mechanical responses and visceral sensitivity.
Interest in guidance molecules, and particularly VEGF, modulating both vascular and neuronal pathology is emerging [
14]. Changes in VEGF levels are associated with alterations in the vascular system of the urinary bladder [
17]. VEGF is increased in bladders of patients with painful bladder syndrome, and this increase is associated with glomerulations on hydrodistension [
24]. However, increased bladder VEGF is not observed in patients who do not show petechial bleeding or in controls [
24], suggesting that VEGF levels are associated with those PBS patients exhibiting alterations in the bladder microvascular system.
At this moment, it is not readily apparent which of the VEGF receptor subtypes mediates the bladder neuroplasticity in the mouse model. Both VEGFR1 and VEGFR2 as well as NRP1 and NRP2 are highly expressed in urothelium and intramural ganglia [
25]. We also reported that control human bladders urothelium present a predominance of VEGFR1 and NRP2 over VEGFR2 and NRP1 immunoreactivity and that PBS patients present a decrease in VEGFR1 and NRP2 expression [
26]. Nevertheless, our results strongly suggest a new and blossoming VEGF-driven processes in the bladder that may be a putative target in neuronal plasticity. However, the role of VEGF pathway in bladder neuroplasticity is in its infancy. In contrast, the roles of NGF and BNDF in neuroplasticity are well established in bladder pathology (e.g., due to spinal cord injury) and have resulted in the testing of NGF-/BNDF-antibodies (or siRNA knockdown) as possible therapeutic options. Therefore, it is tempting to propose that VEGF neutralizing antibodies, such as avastin, or VEGF receptor antagonists may be of benefit to reduce inflammation-induced bladder neuronal plasticity. However, it has to be kept in mind that this growth factor is necessary not only for developing vessels and angiogenesis but also VEGF signaling is required for vascular homeostasis [
27] and the consequences of reduced levels of VEGF can impact the kidney vasculature as seen in pre- eclampsia [
28]. A promising alternative for neutralization of VEGF seems to be the blockade of neuropilins by engineered antibodies [
29]. However, it is too early to predict whether neutralization of neuropilins will have any deleterious effect on the established vasculature.
The rationale for the methodology employed here is based upon our previous observations that VEGF is taken up by the intact urothelium. We showed that following intravesical instillation of a fluorescent VEGF tracer (scVEGF/Cy5.5 ) which only internalizes in cells expressing active VEGF receptors [
30], results in accumulation of this growth factor in suburothelial layers [
31,
32]. After binding these receptors, VEGF may be transcytosed by the urothelial cells into deeper suburothelial layers. Alternatively, VEGF could affect the permeability of the urothelium through mechanisms reminiscent of VEGF’s effects on vascular permeability, which results in paracellular transport of VEGF. Indeed, the protein constituents comprising this highly effective urothelial barrier (tight junctions), occludins [
33], claudins [
34], and zonula occludens-1 [
35] have been recently studied in detail [
36] and are known targets of VEGF-mediated effects on vascular permeability. After the uptake, VEGF produces both bladder inflammation and changes in neuronal plasticity [
17]. The hypothesis that VEGF is taken up by the urothelium was substantiated by the following findings: 1- VEGF receptors are expressed in the mouse [
32] and human bladders [
31]; 2-VEGF neutralizing antibodies significantly reduced inflammation and neuronal plasticity induced by intravesical Bacillus Calmette-Guérin (BCG) stimulation [
17]; 3- An antibody targeting neuropilins (VEGF co-receptors) reduces bladder inflammation [
37]; 4- VEGF itself reproduced the findings obtained with BCG by causing bladder inflammation and sensory nerve plasticity [
17].
How does VEGF produce its effects?
VEGF and its receptors are known neuronal guidance molecules and, therefore, it is expected that they affect nerves. However, the inflammation induced by VEGF may be another possible mechanism leading to an increase in neuronal density. Indeed, VEGF mediates inflammation in the bladder as shown by the findings that instillation of VEGF causes vasodilation, edema, and macrophage recruitment, hallmarks of inflammation [
17], while application of neutralizing VEGF antibodies significantly reduce bladder inflammation [
37]. At this time, there is no definitive evidence suggesting a specific inflammatory cell regulating bladder nerve plasticity. However, given the known trophic effects of VEGF on neurite growth prolonged survival of neurons [
39,
40], and reinnervation following local nerve damage [
41,
42], it is reasonable to propose that inflammatory cells producing VEGF may mediate these growth effects on neurons. This new appreciation of VEGF signaling in bladder inflammation is supported by emerging evidence that VEGF is increased at the site of inflammation, and that infiltrating lymphocytes and other inflammatory cells may represent additional sources of VEGF [
43]. The involvement of cholinergic nerves on bladder inflammatory responses to VEGF suggests a cross-talk between the autonomic and immune systems. Whether the immune system is functionally and anatomically connected to the bladder nervous system remains to be determined. However, a recent investigation proposes that afferent and efferent signals transmitted in the vagus nerve modulate innate immune responses and are components of an inflammatory reflex [
44,
45]. Therefore, it is fair to propose that VEGF increases the cross-talk between the immune and autonomic systems.
VEGF alters bladder function
We showed for the first time that VEGF affects bladder function and modulates micturition reflex pathways.
Analysis of urodynamic parameters recorded in conscious mice confirmed the suggested role of VEGF in modulation of micturition reflex pathways. It is known that exogenous VEGF (or hypoxia induced upregulation of the growth factor) can lead to detrusor and urothelial hypertrophy and hyperplasia [
46]. In addition, previous immunohistochemical analyses of human specimens detected increased innervation in the suburothelial and detrusor layers of the urinary bladder in PBS patients [
47,
48]. In our mouse model, we established that intravesical VEGF treatment resulted in an increase in the density of ChAT fibers in both the detrusor smooth muscle and urothelial layers. This increase in nerve density was associated with altered bladder function as indicated by a decrease in the duration of intermicturition interval, reduced voiding pressure, micturition volume and bladder capacity during continuous filling cystometry. At this time, the individual contributions of sensory and motor nerves to the VEGF-induced increased bladder motility are not clear. Blockade of TRPV1 with capsazepine may shed some light in this respect.
Our results are consistent with other studies which established similar urodynamic changes in animal models of bladder irritation/inflammation [
49‐
51]. For instance, overexpression of neurotrophic nerve growth factor (NGF), a well-known modulator of neural plasticity, in the urinary bladder of mice caused bladder hyperreflexia associated with increased voiding frequency [
7,
52]. These changes were accompanied by an increased density of calcitonin gene-related peptide, SP and neurofilament (NF) 100 positive fibers, as well as tyrosine hydroxylase-positive sympathetic nerve fibers within the suburothelial nerve plexus of the urinary bladder [
7]. Additionally, expression of several TRP channels, including TRPA1, TRPV1, and TRPV4, was increased in the urinary bladder of mice over-expressing NGF [
53]. Interestingly, the urinary bladder phenotype observed in mice with urothelial overexpression of NGF was associated predominantly with the afferent limb of the micturition reflex, whereas our results provide evidence that VEGF affects both afferent and efferent neural pathways. Our data confirmed the suggestion that VEGF may be a potent modulator of neural plasticity in the LUT. Other investigators also suggested that VEGF is a more potent stimulator of neuronal plasticity compared to a number of different neurothrophic factors [
54‐
56]. However, a cross-talk between VEGF and neurotrophins cannot be discarded. On one hand administration of VEGF can support and enhance the growth of regenerating nerve fibers, probably through a combination of angiogenic, neurotrophic, and neuroprotective effects [
57] and conversely, neurotrophins, such as NGF have been described as pro-angiogenic factors [
58]. On the other hand VEGF had neurotrophic effects comparable with BDNF, NT3, or NT4 on the rat isolated pelvic ganglia in culture, [
54]. In addition, VEGF was found to be more potent than BDNF in inducing ChAT-expressing fibers [
54,
55]. Moreover, the synergistic biological activity of VEGF and NGF [
59] is supported by the finding that mechanical stretch of sympathetic neurons seems to induce VEGF expression via a NGF and CNTF signaling pathway [
60]. An intriguing recent hypothesis explaining the cross-talk between VEGF and neurotrophins proposes the convergence of putative signaling downstream of receptor tyrosine kinases [
61]. In this work, Kidins220 /ARMS (Ankyrin repeat-rich membrane spanning) was identified as a main player in the modulation of neurotrophin and VEGF signaling
in vivo, and a primary determinant for neuronal and cardiovascular development [
61]. In support of this hypothesis, it was demonstrated that Kidins220 interacts with neurotrophin, VEGF, ephrin, and glutamate receptors, and is a common downstream target of several trophic stimuli [
61,
62]. Adding to the cross-talk between neurotrophins and VEGF on neuronal plasticity, the present results go one step further by indicating that VEGF alters both sensory (TRPV1) as well as motor (ChAT) nerves. Our present results suggest the idea that across-talk between VEGF and neurothropins controls bladder motor (ChAT) nerve plasticity.
Electrophysiological recordings
in vitro and
in vivo revealed several distinct classes of afferent fibers that participate in transmission of sensory signaling upon physiological bladder filling, noxious distension, chemical irritation and inflammation [
63]. Sensory neurons located within DRG are the first cells to receive afferent input from the pelvic viscera and, therefore, play a substantial role in the development of visceral sensitivity and pelvic discomfort during pathophysiological conditions. DRG neurons express several types of ion channels including TRPV1 and voltage-gated sodium channels (VGSC), both of which are well known transducers of nociceptive processing in pain pathways [
64,
65]. Experiments utilizing animal models of acute and chronic inflammation in the genitourinary tract showed an increased excitability of DRG neurons receiving direct input from the affected organs [
49,
66,
67]. In this study, we determined that instillations of intravesical VEGF caused an up-regulation of VGSC in bladder sensory neurons identified by retrograde labeling. Overexpression of VGSC in bladder DRG cells is associated with increased neuronal excitability and enhanced firing rate [
21]. Our results also support the data from human studies which suggested that abdominal pain and altered bladder and pelvic hypersensitivity in patients with OAB and PBS may involve organizational and/or functional changes in visceral afferent pathways when bladder sensory neurons become sensitized and hyper-responsive to normally innocuous stimuli such as bladder filling [
68,
69].
Multiple sodium channel isoforms are expressed in DRG neurons [
70]. Sodium channels play a central role in neuronal electrogenesis, therefore, variations in the level of expression of any one of the sodium channel isoforms could, in principle, alter their level of excitability [
71]. However, a number of modulatory factors such as neuronal functional status, homeostatic regulation of ion channel expression, post-translational modifications, and interactions with regulating molecules and trophic factors can also significantly affect neuronal excitability [
70]. For instance, brief exposure to NGF, interferon gamma, epidermal growth factor or basic fibroblast growth factor can induce an up-regulation of expression of Na
V1.7 channel [
72]. Interactions of Na
+ channels with partner molecules including NGF [
73,
74], GDNF [
73], contactin [
75,
76], annexin [
77], gabapentin [
78], and other modulators [
79] were established to regulate expression of multiple sodium channel isoforms. Based on these observations, we suggest that the effects of VEGF treatment on Na
+ channels in our study could be associated either with the changes in the expression ratio between different Na
+ channel isoforms in bladder sensory neurons or modulation of Na
+ channel function by regulatory molecules as outlined above [
70]. Additional studies are warranted to identify the exact mechanisms of VEGF action on specific Na
+ channels isoforms and electrical activity of bladder sensory neurons.
The results of behavioral experiments revealed, for the first time, that intravesical VEGF induced the development of abdominal hypersensitivity detected by mechanical stimulation of the lower pelvic region. These effects may be explained, in part, by the ability of VEGF to increase the density of SP and TRPV1 positive fibers [
17]. This suggestion correlates with the previously published studies, which confirmed participation of TRPV1 in the development of abdominal hyperalgesia and neuropathic pain [
80]. Results with TRPV1 knockout mice support the role of TRPV1 in mediating changes in sensitivity. Wang et al. determined that abdominal hyperreactivity and cutaneous allodynia were significantly diminished in these genetically modified animals although the lack of functional TRPV1 receptors did not improve the histological changes in the inflamed bladder induced by either cyclophosphamide (CYP) or acrolein [
51]. In addition to the involvement of TRPV1 afferents in pelvic sensitivity, other receptors and molecules can also contribute to abdominal hyperalgesia depending on the model and nature of chosen inflammatory agents [
81]. Thus, increased peripheral sensitivity in mice with bacterial cystitis was related to activation of toll-like receptor 4 [
82]. In the acrolein model of bladder inflammation in rats, increased mechanical sensitivity was conveyed, in part, via NGF and trk receptors [
83]. Likewise, inflammatory events experienced earlier in life were established to trigger long lasting changes in sensory pathways leading to altered pelvic sensations during the adulthood [
84,
85]. Altogether, our data provide direct evidence that VEGF-induced neurogenic inflammation in the urinary bladder is associated with significant structural and functional changes that may play a key role in the development of neurogenic bladder dysfunctions in humans.