Expression of the VEGF and angiopoietin genes in endometrial atypical hyperplasia and endometrial cancer

C M Holland, K Day, A Evans, S K Smith, C M Holland, K Day, A Evans, S K Smith

Abstract

Angiogenesis is critical for the growth and metastasis of endometrial cancer and is therefore an important therapeutic target. Vascular endothelial growth factor-A (VEGF-A) is a key molecule in angiogenesis, but the identification of related molecules and the angiopoietins suggests a more complex picture. We investigated the presence of transcripts for VEGF-A, VEGF-B, VEGF-C, VEGF-D, Angiopoietin-1 and Angiopoietin-2 in benign endometrium, atypical complex hyperplasia (ACH) and endometrioid endometrial carcinoma using in situ hybridisation. We confirmed the presence of VEGF-A mRNA in the epithelial cells of cancers examined (13 out of 13), but not in benign endometrium or ACH. We also demonstrate, using quantitative polymerase chain reaction, that levels of VEGF-B mRNA are significantly lower in endometrial cancer than benign endometrium. We conclude that loss of VEGF-B may contribute to the development of endometrial carcinoma by modulating availability of receptors for VEGF-A.

Figures

Figure 1
Figure 1
Reverse transcription PCR analysis of VEGF-C in samples of benign endometrium (A–G), ACH (H–J) and endometrioid endometrial carcinoma. Well differentiated carcinoma (K–M), moderately differentiated carcinoma (N–S) and poorly differentiated carcinoma (T–W). Lane r represents an RNA control in which the RT step was omitted and lane c represents a negative control in which the cDNA was replaced by water. The size of the transcripts (shown on the right of the figure) was estimated by co-electrophoresis of a 1 kb ladder (lane l).
Figure 2
Figure 2
Expression of mRNA encoding VEGF-related factors in the epithelial cells of endometrioid endometrial carcinoma and ACH. In situ hybridisation with VEGF-A antisense probe in a moderately differentiated (FIGO grade 2) carcinoma shown under dark-field (A) and light-field (C) conditions shows hybridisation in epithelial carcinoma cells. There is no specific hybridisation when a sense probe (negative control) is used (B). The scale bar in (B) applies to (A), (B), (E), (F), (I) and (J). Immunostaining of a serial section with anti-human cytokeratin confirms the epithelial localisation of silver grains (D) (scale bar applies to ACH (C) and (D). In situ hybridisation with VEGF-B antisense probe in ACH shown under dark-field (E) and light-field (G) conditions shows diffuse hybridisation in epithelial and stromal cells. There is no specific hybridisation when a sense probe (negative control) is used (F). Immunostaining with anti-human cytokeratin (H) identifies the epithelial and stromal cells (H) (scale bar applies to (G) and (H). In situ hybridisation with VEGF-C antisense probe in a poorly differentiated (FIGO grade 3) carcinoma shown under dark-field conditions (I) shows no specific hybridisation when compared with a human B-cell lymphoma (J).
Figure 3
Figure 3
Expression of mRNA encoding VEGF-D and Ang-2 in macrophages infiltrating a poorly differentiated (FIGO grade 3) endometrioid endometrial carcinoma. In situ hybridisation with VEGF-D antisense probe shown under dark-field (A) conditions and light-field conditions (C). In situ hybridisation with VEGF-D sense (control) probe shows no specific hybridisation (B) (scale bar applies to (A), (B), (E) and (F)). Immunostaining with anti-human CD68 in a serial section (D), localises the silver grains to tumour associated macrophages (TAMs) (scale bar applies to (C), (D), (G) and (H)). In situ hybridisation with Ang-2 antisense probe shown under dark-field (E) and light-field (G) conditions. In situ hybridisation with Ang-2 sense (control) probe (F) shows no specific hybridisation. Anti-human cytokeratin staining (H) localises the hybridisation to TAMs.
Figure 4
Figure 4
Quantitative PCR for gene transcripts in benign and malignant endometrium. Levels of gene transcript are shown normalised to an endogenous control sequence. Transcript levels are expressed as arbitrary units and represent the mean for each histological group. Experiments for each sample were performed in triplicate. (A) The level of mRNA encoding VEGF-B is generally higher in benign postmenopausal endometrium than in CAH (P=0.13) and endometrial cancer (P=0.04). (B) mRNA encoding Ang-2 is generally present at higher levels in endometrial cancer than in benign postmenopausal endometrium.

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