Therapeutic effects of axitinib, an anti-angiogenic tyrosine kinase inhibitor, on interstitial cystitis

Abstract

To investigate the therapeutic effects of axitinib, a tyrosine kinase inhibitor, in an interstitial cystitis (IC) rat model. IC patients with or without Hunner lesion and non-IC controls were enrolled (n = 5/group). Bladder tissues were stained with vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), platelet-derived growth factor (PDGF), and PDGF receptor B (PDGFR-B). The IC group showed extensive VEGFR-2 and PDGFR-B staining compared with controls. Next, ten-week-old female Sprague Dawley rats were divided into three groups (n = 10/group): sham, hydrochloride (HCl), and axitinib groups. One week after HCl instillation (day 0), the axitinib group received oral axitinib (1 mg/kg) for five consecutive days and pain was evaluated daily. Bladder function, histology and genetics were evaluated on day 7. The pain threshold significantly improved 3 days after axitinib administration. Axitinib decreased non-voiding contraction and increased the micturition interval and micturition volume and alleviated urothelial denudation, angiogenesis, mast cell infiltration, and fibrosis. HCl instillation increased the expression of tyrosine kinase receptors, including VEGFR-2 and PDGFR-B; axitinib administration inhibited their expression. Oral administration of axitinib improved pain, voiding profiles, and urothelial integrity by inhibiting angiogenesis in IC rat model. Axitinib may have potential therapeutic efficacy in IC patients.

Figure 1

VEGFR-2 and PDGFR-B expression was increased in bladder tissues from IC patients. (A) Percentage of positive staining area and (B) representative images of each immunohistochemical staining. All data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the BPS group and #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the control with one-way ANOVA.

Figure 2

Axitinib increased the withdrawal threshold in hydrochloride (HCl)-induced interstitial cystitis (IC) rats. The withdrawal thresholds (pain score) of sham, HCl-induced IC, and axitinib-treated groups were compared using the manual von Frey test (up-down method). All data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the HCl group with one-way ANOVA with Bonferroni post-tests.

Figure 3

Axitinib restored bladder dysfunction in hydrochloride (HCl)-induced interstitial cystitis (IC) rats. (A) Representative awake cystometry results and (B) quantitative analysis of voiding parameters 2 weeks after HCl or saline instillation into rat bladders. All quantitative data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the HCl group with one-way ANOVA with Bonferroni post-tests. (IVP; intravesical pressure, IAP; intra-abdominal pressure).

Figure 4

Axitinib reduced histological changes in hydrochloride (HCl)-induced interstitial cystitis (IC) rat bladders. (A) Representative images of cytokeratin staining and quantification of urothelial denudation (magnification × 100, scale bar = 200 μm). (B) Representative images of CD31 staining and quantification of angiogenesis (magnification × 200, scale bar = 200 μm). (C) Toluidine blue staining and quantification of mast cell infiltration (magnification × 100, scale bar = 200 μm). (D) Representative images for Masson’s trichrome staining (magnification × 200, scale bar = 200 μm). All quantitative data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the HCl group with one-way ANOVA with Bonferroni post-tests.

Figure 5

Effect of axitinib therapy on the expression of genes related to the pathogenesis of hydrochloride (HCl)-induced interstitial cystitis (IC) in rat bladders. Real-time quantitative polymerase chain reaction analysis of platelet-derived growth factor (PDGF) related genes (A), and vascular endothelial growth factor (VEGF)-related genes (B) in the indicated bladder tissues. Expression is presented as % GAPDH expression and shown as a dot plot of the mean and SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the HCl group with one-way ANOVA with Bonferroni post-tests. (C,D) Fluorescent immunohistochemical detection of VEGFR2 and PDGFR-α protein (green) in the indicated bladder tissues (magnification × 400). Nuclei were stained with 4’,6-diamino-2-phenylindole (blue). Quantitative data of each staining are presented on the right side of the indicated representative pictures. All quantitative data were normalized to those of the sham group and are presented as the mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the HCl-IC with the Bonferroni post-test.