Anti-angiogenic therapy as potential treatment for adenomyosis

Abstract

Adenomyosis is characterized by abnormal uterine bleeding, dysmenorrhea and subfertility. Increased expression of angiogenesis markers in adenomyosis presents a treatment opportunity and was studied in an adenomyosis mouse model. Mice were administered tamoxifen (1 mg/kg) on neonatal days 2-5. At six weeks of age, mice received oral treatment with axitinib 3 mg/kg ('dose I/AX3', n = 34), axitinib 25 mg/kg ('dose II/AX25' n = 34), or with vehicle-only ('placebo', n = 34). The prevalence and severity of adenomyosis were assessed. An adenomyosis severity index was calculated by multiplying mean grade/mouse by the percentage affected surface area. Angiogenesis-related gene expression was evaluated using real-time quantitative PCR. 101 mice completed adenomyosis induction and could be analyzed. The prevalence of adenomyosis was 30/33 (90.0%) in dose I, 29/34 (85.3%) in dose II, and 30/34 (88.2%) in placebo treated mice (p = 0.78). High grade (2/3) adenomyosis was significantly less prevalent in mice treated with axitinib dose II (n = 19, 55.9%) than in the placebo group (n = 27, 79.4%, p < 0.05). The adenomyosis severity index was reduced by 48% in the axitinib-treated groups (dose I, p < 0.05). The expression of angiogenic growth factors was reduced in the dose I and II axitinib-treated groups compared to the placebo-treated group. Following these promising first results, further research should focus on commonality among different angiostatic drugs, potential side effects, as well as the method and timing of application.

Keywords: Adenomyosis; Angiogenesis; Anti-angiogenic agent; Axitinib; CD-1 mice; Tamoxifen.

Fig. 1

Schematic overview of experimental groups and timeline for inducing adenomyosis in CD1 mice using tamoxifen, followed by treatment with axitinib 3 mg/kg (dose I) or axitinib 25 mg/kg (dose II) from week 6 until week 9. After termination of the mice at week 9, all uteri were analyzed for the presence of adenomyosis

Fig. 2

Adenomyosis severity index explained. The grade of adenomyosis (H&E) was multiplied by the surface area of vimentin stained adenomyosis per transverse uterus section. The adenomyosis severity index is the average of all sections analyzed per uterine horn. Avg. Average, H&E hematoxylin & eosin

Fig. 3

Representative images of HE (top row) and α-SMA (bottom row) stained transverse sections of mice uteri after neonatal treatment with vehicle (left column) and tamoxifen (right column). The uteri of vehicle treated mice show no signs of ectopic endometrium in the myometrium (Grade 0) on the HE stained slice, and an intact myometrium on the α-SMA stained slice, while the uteri of tamoxifen treated mice demonstrate ectopic endometrium (black arrow) in the myometrium (Grade 2 presented) on the HE stained slice, and an interrupted myometrium on the α-SMA stained slice with thin residual unaffected myometrium

Fig. 4

A Bar chart of the prevalence (count) of Grade 0/1/2/3 adenomyosis on the HE stained mice uteri, per treatment group. B, Proportion of low grade versus high grade adenomyosis per treatment group. C Adenomyosis severity score (mean grade * mean percentage surface area of adenomyosis per mouse uterus specimen) per treatment group. Mean grade of adenomyosis of all transverse sections per mouse uterus per treatment group. D Mean grade of adenomyosis of all transverse sections per mouse uterus, per staining method and per treatment group. Data are shown as median ± range for all mice pooled. * significance p < 0.05, ** p < 0.001. Treatment groups: placebo (CMC, n = 34), AX3 (axitinib 3 mg/kg, n = 33) and AX25 (axitinib 25 mg/kg, n = 34)

Fig. 5

Top: From left to right the HE, α-SMA and Vimentin staining results of grade I adenomyosis in transverse sections of mice uteri. Bottom: from left to right the HE, α-SMA and Vimentin staining results of grade II adenomyosis in transverse sections of mice uteri. Black arrow indicates the invaded ectopic endometrium with thin residual unaffected myometrium in each uterine transverse section

Fig. 6

Microvascular density per unit of measurement in the eutopic endometrium (A), ectopic endometrium (B), and myometrium (C) of mice from the validation experiment treated with vehicle (Healthy control n = 6) or tamoxifen (ADM, n = 6) and sacrificed at 6 weeks of age, and the mice with tamoxifen-induced adenomyosis treated with axitinib 25 mg/kg (Ax25, n = 34), axitinib 3 mg/kg (Ax3, n = 34), or placebo (ADM, n = 34) sacrificed at 9 weeks of age. Data shown as median with interquartile range. * significance p < 0.05, ** p < 0.001

Fig. 7

Real-time (RT)-PCR molecular profiling of the mouse uteri treated with CMC, axitinib dose I (AX3 = 3 mg/kg), or axitinib dose II (AX25 = 25 mg/kg). Expression fold change of angiogenesis-related genes plGF, VEGF-R1, VEGF-R2, VEGF-R3, VEGF-A, VEGF-B, CD31 and Collagen determined by 2^- ΔΔCt of RT-PCR. Mean relative expressions are shown as mean with the SEM. CMC, n = 7; AX3, n = 6; AX25, n = 7. *p < 0.05 and **p < 0.001