Limits...
Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes

View Article: PubMed Central - PubMed

ABSTRACT

Background: Angiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice.

Methods: Cycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment.

Results: ABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning.

Conclusion: Our findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.

Electronic supplementary material: The online version of this article (doi:10.1186/s12958-016-0192-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


ABT-898 does not affect the estrous cycle in female mice. Representative vaginal cytology of the estrous cycle in mice dosed with ABT-898 (a–e) or 5 % dextrose (f–j). Keratinized epithelial cells (a, f) indicative of estrous were used to denote the start of a new estrous cycle. The number of estrous cycles did not significantly differ between ABT-898 and 5 % dextrose groups (3.6 vs 3.3, p = 0.561) over the course of the 21 day dosing period (k). Female reproductive tracts from mice dosed with ABT-898 (l) or 5 % dextrose (m). Gross observations indicated that female reproductive tracts from ABT-898 (l) treated mice did not differ from 5 % dextrose control in size (m), morphology or vascularity. Images (a–j) are magnified 400× and scale bars represent 75 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5015213&req=5

Fig1: ABT-898 does not affect the estrous cycle in female mice. Representative vaginal cytology of the estrous cycle in mice dosed with ABT-898 (a–e) or 5 % dextrose (f–j). Keratinized epithelial cells (a, f) indicative of estrous were used to denote the start of a new estrous cycle. The number of estrous cycles did not significantly differ between ABT-898 and 5 % dextrose groups (3.6 vs 3.3, p = 0.561) over the course of the 21 day dosing period (k). Female reproductive tracts from mice dosed with ABT-898 (l) or 5 % dextrose (m). Gross observations indicated that female reproductive tracts from ABT-898 (l) treated mice did not differ from 5 % dextrose control in size (m), morphology or vascularity. Images (a–j) are magnified 400× and scale bars represent 75 μm

Mentions: Prior to the study, to confirm that each lot of the thrombospondin-1 mimetic peptide ABT-898 had anti-angiogenic properties, we performed in vitro angiogenesis assays as previously described [13]. ABT-898 was able to completely ameliorate endothelial cell tube formation in vitro (data not shown). Non-pregnant cycling female mice were treated for 21 consecutive days with ABT-898 at a concentration (100 mg/kg) that had previously been shown to reduce neovascularization of endometriosis lesions [13], or 5 % dextrose control. Vaginal cytology was analyzed each day to assess the effect of ABT-898 on the estrous cycle. Mice showed the classical stages of the estrous cycle irrespective of treatment (representative vaginal cytology for ABT-898 Fig. 1a–e, 5 % dextrose control 1 F–J). The total number of estrous cycles did not differ between ABT-898 treated (3.75 cycles) and 5 % dextrose control groups (3.3 cycles, Fig. 1k, p = 0.561).Fig. 1


Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes
ABT-898 does not affect the estrous cycle in female mice. Representative vaginal cytology of the estrous cycle in mice dosed with ABT-898 (a–e) or 5 % dextrose (f–j). Keratinized epithelial cells (a, f) indicative of estrous were used to denote the start of a new estrous cycle. The number of estrous cycles did not significantly differ between ABT-898 and 5 % dextrose groups (3.6 vs 3.3, p = 0.561) over the course of the 21 day dosing period (k). Female reproductive tracts from mice dosed with ABT-898 (l) or 5 % dextrose (m). Gross observations indicated that female reproductive tracts from ABT-898 (l) treated mice did not differ from 5 % dextrose control in size (m), morphology or vascularity. Images (a–j) are magnified 400× and scale bars represent 75 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015213&req=5

Fig1: ABT-898 does not affect the estrous cycle in female mice. Representative vaginal cytology of the estrous cycle in mice dosed with ABT-898 (a–e) or 5 % dextrose (f–j). Keratinized epithelial cells (a, f) indicative of estrous were used to denote the start of a new estrous cycle. The number of estrous cycles did not significantly differ between ABT-898 and 5 % dextrose groups (3.6 vs 3.3, p = 0.561) over the course of the 21 day dosing period (k). Female reproductive tracts from mice dosed with ABT-898 (l) or 5 % dextrose (m). Gross observations indicated that female reproductive tracts from ABT-898 (l) treated mice did not differ from 5 % dextrose control in size (m), morphology or vascularity. Images (a–j) are magnified 400× and scale bars represent 75 μm
Mentions: Prior to the study, to confirm that each lot of the thrombospondin-1 mimetic peptide ABT-898 had anti-angiogenic properties, we performed in vitro angiogenesis assays as previously described [13]. ABT-898 was able to completely ameliorate endothelial cell tube formation in vitro (data not shown). Non-pregnant cycling female mice were treated for 21 consecutive days with ABT-898 at a concentration (100 mg/kg) that had previously been shown to reduce neovascularization of endometriosis lesions [13], or 5 % dextrose control. Vaginal cytology was analyzed each day to assess the effect of ABT-898 on the estrous cycle. Mice showed the classical stages of the estrous cycle irrespective of treatment (representative vaginal cytology for ABT-898 Fig. 1a–e, 5 % dextrose control 1 F–J). The total number of estrous cycles did not differ between ABT-898 treated (3.75 cycles) and 5 % dextrose control groups (3.3 cycles, Fig. 1k, p = 0.561).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Angiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice.

Methods: Cycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment.

Results: ABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning.

Conclusion: Our findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.

Electronic supplementary material: The online version of this article (doi:10.1186/s12958-016-0192-7) contains supplementary material, which is available to authorized users.

No MeSH data available.