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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.


CD31 and vimentin immunohistochemistry of ABT-898 treated and 5 % dextrose control uteri and ovaries. Semi-quantitative analysis of CD31+ blood vessels in the endometrium found no significant difference (p = 0.45, i) between ABT-898 (a) and 5 % dextrose (b) groups. Immunohistochemistry for CD31 also found that blood vessel development around antral follicles was not altered by ABT-898 (c, f, d, g), and semi-quantitative analysis found no significant difference (p = 0.75). Vimentin immunohistochemistry showed no difference in structure or number of microvessels (green arrows) between ABT-898 (e) and 5 % dextrose (h) corpus luteum. Scale bars represent 75 μm. FOV: Field of view
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Fig3: CD31 and vimentin immunohistochemistry of ABT-898 treated and 5 % dextrose control uteri and ovaries. Semi-quantitative analysis of CD31+ blood vessels in the endometrium found no significant difference (p = 0.45, i) between ABT-898 (a) and 5 % dextrose (b) groups. Immunohistochemistry for CD31 also found that blood vessel development around antral follicles was not altered by ABT-898 (c, f, d, g), and semi-quantitative analysis found no significant difference (p = 0.75). Vimentin immunohistochemistry showed no difference in structure or number of microvessels (green arrows) between ABT-898 (e) and 5 % dextrose (h) corpus luteum. Scale bars represent 75 μm. FOV: Field of view

Mentions: We next investigated whether ABT-898 would reduce the blood vessel density, or alter endothelial cell structure in organs where physiological angiogenesis occurs, the uterus and the ovary. We performed immunohistochemistry for CD31, a pan endothelial cell marker, on ovarian and uterine sections. Semi-quantitative analysis of uterine sections found no difference in the number of CD31 (+) blood vessels between ABT-898 (Fig. 3a) and control (Fig. 3b) groups (p = 0.65, Fig. 3i). We also analyzed CD31 (+) blood vessels in antral follicles, and found no difference in the number or structure of blood vessels from ABT-898 (Fig. 3c, d) compared to control (Fig. 3f, g) groups (p = 0.803, Fig. 3i). Immunohistochemistry for vimentin, an intermediate filament expressed in cells of mesenchymal origin, was used to investigate corpora lutea. Qualitative observations found no difference in the size and structure of the corpora lutea between ABT-898 (Fig. 3e) and control (Fig. 3h) groups. Although vimentin is expressed in other cell types of the corpus luteum besides endothelial cells, blood vessels are easily identified using this stain and the numbers were similar between ABT-898 (Fig. 3e) and control (Fig. 3h) groups.Fig. 3


Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes
CD31 and vimentin immunohistochemistry of ABT-898 treated and 5 % dextrose control uteri and ovaries. Semi-quantitative analysis of CD31+ blood vessels in the endometrium found no significant difference (p = 0.45, i) between ABT-898 (a) and 5 % dextrose (b) groups. Immunohistochemistry for CD31 also found that blood vessel development around antral follicles was not altered by ABT-898 (c, f, d, g), and semi-quantitative analysis found no significant difference (p = 0.75). Vimentin immunohistochemistry showed no difference in structure or number of microvessels (green arrows) between ABT-898 (e) and 5 % dextrose (h) corpus luteum. Scale bars represent 75 μm. FOV: Field of view
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: CD31 and vimentin immunohistochemistry of ABT-898 treated and 5 % dextrose control uteri and ovaries. Semi-quantitative analysis of CD31+ blood vessels in the endometrium found no significant difference (p = 0.45, i) between ABT-898 (a) and 5 % dextrose (b) groups. Immunohistochemistry for CD31 also found that blood vessel development around antral follicles was not altered by ABT-898 (c, f, d, g), and semi-quantitative analysis found no significant difference (p = 0.75). Vimentin immunohistochemistry showed no difference in structure or number of microvessels (green arrows) between ABT-898 (e) and 5 % dextrose (h) corpus luteum. Scale bars represent 75 μm. FOV: Field of view
Mentions: We next investigated whether ABT-898 would reduce the blood vessel density, or alter endothelial cell structure in organs where physiological angiogenesis occurs, the uterus and the ovary. We performed immunohistochemistry for CD31, a pan endothelial cell marker, on ovarian and uterine sections. Semi-quantitative analysis of uterine sections found no difference in the number of CD31 (+) blood vessels between ABT-898 (Fig. 3a) and control (Fig. 3b) groups (p = 0.65, Fig. 3i). We also analyzed CD31 (+) blood vessels in antral follicles, and found no difference in the number or structure of blood vessels from ABT-898 (Fig. 3c, d) compared to control (Fig. 3f, g) groups (p = 0.803, Fig. 3i). Immunohistochemistry for vimentin, an intermediate filament expressed in cells of mesenchymal origin, was used to investigate corpora lutea. Qualitative observations found no difference in the size and structure of the corpora lutea between ABT-898 (Fig. 3e) and control (Fig. 3h) groups. Although vimentin is expressed in other cell types of the corpus luteum besides endothelial cells, blood vessels are easily identified using this stain and the numbers were similar between ABT-898 (Fig. 3e) and control (Fig. 3h) groups.Fig. 3

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.