VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG

Abstract

Background: This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity.

Methods: Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (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 neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed 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.

Figure 1

Mouse bladder inflammation. Representative photomicrographs from the urinary bladders isolated from mice chronically treated with 4 weekly instillations of PBS (A), BCG (B), VEGF₁₆₅ (C), and VEGF₁₂₁ (D). Black box inserts on B, C, and D correspond to a 200% magnification of the original dashed box area of the respective figures and illustrate inflammatory cells. White transversal bars indicate the urothelium, yellow bars indicate the sub-urothelium, and green bars indicate the detrusor smooth muscle. Green arrow indicates perivascular infiltrate of inflammatory cells (D). See Figures 2 and 3 for quantification of inflammatory cells by image analysis.

Figure 2

Representative photomicrographs of double immunofluorescence (MPO and F4/80) of the urinary bladder mucosa isolated from mice that received 4 weekly instillations of BCG (A and D), VEGF₁₆₅ (B and E), and VEGF₁₂₁ (C and F). Qualitatively all stimuli induced the migration of both F4/80+ macrophages and MPO+ neutrophils. Quantification of the results is being present in Figures 3 and 4 and summarized on Table 1. D, E, and F correspond to magnification of the original dashed box area of the respective figures A, B, and C.

Figure 3

Chronic BCG instillation induces a predominant migration of myeloperoxidase-positive neutrophils and, to a lesser extent, F4/80-positive macrophages that are blunted by co-treatment with VEGF neutralizing antibody (B20). Quantification of myeloperoxidase-IR (A-B) and F4/80-IR (C-D) in the sub urothelium and detrusor muscle of bladders isolated from: control (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of PBS), BCG-treated (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of BCG), and B20-treated (mice receiving i.p. B20 [5 mg/kg; twice a week] concomitant to 4 weekly instillations of BCG). N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 4

VEGF induces bladder inflammation. Quantification of myeloperoxidase-IR (A-B) and F4/80-IR (C-D) in the sub urothelium and detrusor muscle of bladders isolated from: 4 weekly instillations with PBS, single instillation with VEGF₁₆₅, 4 weekly instillations with VEGF₁₆₅, 6 weekly instillations with VEGF₁₆₅, a single instillation with VEGF₁₂₁, and 4 weekly instillations of VEGF₁₂₁. Mice were euthanized one week after the last instillation and the urinary bladders removed for IF. N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 5

TRPV1-IR in the mouse urinary bladder. Representative photomicrograph illustrating distribution of TRPV1-IR in the urinary bladder isolated from C57BL/6 mice receiving chronic PBS instillation (A) shows TRPV1-IR extending from the base (cyan arrow) of the urothelium (white transversal bar) towards the sub-urothelium (yellow transversal bar). B) No immunostaining was observed in bladders isolated from TRPV1^-/- mice indicating that this antibody specifically identifies TRPV1-IR nerve fibers. C indicates some TRPV1-positive nerves crossing the urothelial layer (white arrows) and D illustrates TRPV1-positive nerves surrounding urothelial cells. White waved line on D highlights the bladder lumen.

Figure 6

Mouse bladder nerves. Representative photomicrographs illustrating distribution of IR for: TRPV1 (A-B), CGRP (C-D), PGP9.5 (E-F), and SP (G-H) in urinary bladders isolated from C57BL6 mice that received chronic PBS instillation (left panels) or chronic BCG instillation (right panels). White transversal bars indicate the urothelium, yellow bars indicate the sub-urothelium, and green bars indicate the detrusor smooth muscle. Yellow arrows indicate non-specific labeling of urothelial cells with PGP9.5 (F) and CGRP (C). See Figure 6 for quantification of IR by image analysis.

Figure 7

Chronic BCG instillation increases IR for the pan neuronal marker, PGP9.5, and sensory nerve markers, TRPV1, SP, and CGRP. Quantification of PGP9.5- TRVP1-, SP-, and CGRP-IR in the sub urothelium (A) and detrusor muscle (B) of bladders isolated from: control (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of PBS) and BCG-inflamed mice (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of BCG). N = 8 per group. Statistical significance was set for p values < 0.05. NS = non significant.

Figure 8

VEGF mediates chronic BCG-induced bladder nerve plasticity. Quantification of PGP9.5-IR (A-B) and TRPV1-IR (C-D) in the urinary bladder isolated from: control (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of PBS), BCG-treated (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of BCG), and B20-treated (mice receiving B20 [twice a week] concomitant to 4 weekly instillations of BCG). N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 9

Effect of NRP1 and NRP2 blocking antibodies on BCG-induced bladder nerve plasticity. Quantification of PGP9.5-IR (A-B) and TRPV1-IR (C-D) in the urinary bladder isolated from: control (mice receiving i.p. PBS [twice a week] concomitantly with 4 weekly instillations of PBS), BCG-treated (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of BCG), NRP1^A-BCG treated (mice receiving NRP1^A [twice a week] concomitant to 4 weekly instillations of BCG), NRP1^B-BCG treated (mice receiving NRP1^B [twice a week] concomitant to 4 weekly instillations of BCG), NRP2-BCG treated (mice receiving NRP2^B [twice a week] concomitant to 4 weekly instillations of BCG), and NRP2-PBS treated (mice receiving NRP2^B [twice a week] concomitant to 4 weekly instillations of PBS). N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 10

Effect of NRP2 blocking antibodies on BCG-induced CGRP- and SP-IR. Quantification of CGRP-IR (A-B) and SP-IR (C-D) in the urinary bladder isolated from: control (mice receiving i.p. PBS [twice a week] concomitant to 4 weekly instillations of PBS), BCG-treated (mice receiving i.p. PBS [twice a week] concomitantly with 4 weekly instillations of BCG), and NRP2-BCG treated (mice receiving NRP2^B [twice a week] concomitant to 4 weekly instillations of BCG). N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 11

VEGF₁₆₅ and VEGF₁₂₁ recapitulate BCG-induced bladder nerve plasticity. Quantification of PGP9.5-IR (A-B) and TRPV1-IR (C-D) in the urinary bladder isolated 1 week after: 4 weekly instillations with PBS, single instillation with VEGF₁₆₅, 4 weekly instillations with VEGF₁₆₅, 6 weekly instillations with VEGF₁₆₅, a single instillation with VEGF₁₂₁, and 4 weekly instillations of VEGF₁₂₁. N = 8 per group. Statistical significance was set for p values < 0.05.

Figure 12

Representative photomicrographs of double immunofluorescence (CD31 and KI67) of the urinary bladder mucosa isolated from mice instilled chronically (4 weekly instillations) of: BCG, VEGF₁₆₅, and VEGF₁₂₁. The area of CD31+ blood vessels was used to calculated microvessel density (Figure 13A) and the co-localization between CD31+ endothelial cells and KI67 expression was used to calculate the degree of angiogenesis (Figure 13 B).

Figure 13

Effects of instillation of BCG, VEGF₁₆₅, and VEGF₁₂₁ into the urinary bladder resulted in alterations of vascular plasticity. (A) Microvessel density (MVD) was determined by computing the area occupied by CD31+ blood vessels that was expressed as percent of the cross-sectional area. (B) Angiogenesis was defined by the proliferation of CD31+ blood vessels. For this purpose, the expression of a marker of proliferation (KI67) was calculated taking CD31+ blood vessels as region of interest (ROI).