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

BACKGROUND: It 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. METHODS: We 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. RESULTS: Lymphatic 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. CONCLUSIONS: These results illustrate that VEGF-D over-expression has differential effects on the uterine vasculature. These effects may facilitate VEGF-D's ability to promote endometrial cancer metastasis and disease progression

In situ Biological Dose Mapping Estimates the Radiation Burden Delivered to ‘Spared’ Tissue between Synchrotron X-Ray Microbeam Radiotherapy Tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to ‘spared’ tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30–40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies

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

Increased blood and lymphatic vessel area after progestin exposure.

<p>Blood and lymphatic vessel cross sectional areas are increased in endometrium from women treated with LNG-IUS prior to hysterectomy for heavy menstrual bleeding. (A) The mean area of the five largest blood vessels in the functional layer and the basal layer in untreated control (white bars) and LNG-IUS treated (grey bars) endometrium. (B) The mean area of the five largest lymphatic vessels in the functional layer and the basal layer of untreated control (white bars) and LNG-IUS treated (grey bars) endometrium. Columns represent means ± SE. *, <i>P</i>&lt;0.05.</p

VEGF-D protein in endometrial decidual cells.

<p>Western analysis of vascular endothelial cell growth factor-C (VEGF-C) and VEGF-D peptide expression in primary cultures of human endometrial stromal cells (HESC). Panels A and B show representative data from 3 samples. Panels C and D show combined densitometry data from all 5 samples. (A) VEGF-C peptide expression (58, 41, 29/31 and 21 kD) in HESC (ND) and decidualised HESC (Dec). (B) VEGF-D peptide expression (53, 41, 29/31 and 21 kD) in HESC and decidualised HESC. (C–D) Densitometry of VEGF-C and VEGF-D peptide expression. White bars representing control non-decidualised HESC and black bars representing decidualised HESC. Columns represent means ± SE; * <i>P</i>&lt;0.05.</p

VEGF-D immunostaining in endometrial decidual cells.

<p>Distinct VEGF-D immunostaining (brown) is present in pre-decidual cells of endometrium from women treated with LNG-IUS prior to hysterectomy. (A) Micrograph illustrating difference in VEGF-D immunostaining (brown) intensity in stromal pre-decidualised cells (pd) compared to non-decidualised cells (nd). (B) Higher power image of pre-decidualised stromal cells showing VEGF-D immunostaining in brown. (C) Higher power image of non-decidualised stromal cells showing reduced VEGF-D immunostaining. (D) Isotype matched negative control. Black arrow: blood vessel. (sections are lightly counterstained with haematoxylin to identify cell nuclei in blue).</p

VEGF-D immunostaining of xenograft decidual cells.

<p>Representative micrographs of human endometrial xenografts from NOD/scid mice treated with estradiol valerate (A,B) or medroxyprogesterone acetate (C,D). Serial sections were stained with haematoxylin and eosin (A,C) or immunostained with VEGF-D (B,D). Inset in (B): Isotype matched negative control. Note the pre-decidual cells in MPA-treated sections. ep: epithelium, g: glands, pd: pre-decidual stroma, s: stroma.</p

Endometrial blood and lymphatic vessel density after progestin exposure.

<p>Blood and lymphatic vessel densities do not change in endometrium from women treated with LNG-IUS prior to hysterectomy for heavy menstrual bleeding. (A) Vascular density of blood vessels in untreated control (white bars) and LNG-IUS treated (grey bars) samples of the functional layer and the basal layer. (B) Lymphatic vessel density in untreated and LNG-IUS treated endometrial samples. Columns represent means ± SE. *, <i>P</i>&lt;0.05.</p