Fiona L. Lederman

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti‐VEGF antibodies indicated that other angiogenic factors may be involved. VEGF‐D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor‐2 (VEGFR‐2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor‐binding domain of VEGF‐D and the use of these antibodies to localize VEGF‐D in malignant melanoma. VEGF‐D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF‐D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF‐D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR‐2 and VEGFR‐3. As VEGF receptors can be up‐regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF‐D in smooth muscle of the blood vessels suggest that VEGF‐D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells. Copyright

Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer.

BackgroundIt has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression.MethodsWe initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3) and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells.ResultsLymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium), although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating endothelial cells, and the cross sectional area of vessel profiles were significantly increased in response to VEGF-D in comparison to control tumor cells. In contrast, no significant changes were noted in myometrial blood vessels. In addition, examples of invading cells or tumor emboli were observed in mice receiving VEGF-D expressing 293EBNA cells.ConclusionsThese results illustrate that VEGF-D over-expression has differential effects on the uterine vasculature. These effects may facilitate VEGF-Ds ability to promote endometrial cancer metastasis and disease progression.

Dilated Thin-Walled Blood and Lymphatic Vessels in Human Endometrium: A Potential Role for VEGF-D in Progestin-Induced Break-Through Bleeding

Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders. Episodes of irregular endometrial bleeding or breakthrough bleeding (BTB) are a major unwanted side effect of progestin treatment, such that BTB is the leading cause for discontinued use of an otherwise effective and popular medication. The cellular mechanisms leading to BTB are poorly understood. In this study, we make the novel finding that the large, dilated, thin walled vessels characteristic of human progestin-treated endometrium include both blood and lymphatic vessels. Increased blood and lymphatic vessel diameter are features of VEGF-D action in other tissues and we show by immunolocalisation and Western blotting that stromal cell decidualisation results in a significant increase in VEGF-D protein production, particularly of the proteolytically processed 21 kD form. Using a NOD/scid mouse model with xenografted human endometrium we were able to show that progestin treatment causes decidualisation, VEGF-D production and endometrial vessel dilation. Our results lead to a novel hypothesis to explain BTB, with stromal cell decidualisation rather than progestin treatment per se being the proposed causative event, and VEGF-D being the proposed effector agent.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus.

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin bA subunit (Inhba) and the inhibin a subunit (Inha) in uterine tissues during early pregnancy (days 1–4, preimplantation) and in response to exogenous 17b-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17b-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus