Localization and functional study of VEGF receptors in normal and adenomatous pituitary: evidence for a non-angiogenic role of VEGF

The role of members of the Vascular Endothelial Growth Factor (VEGF) family and their receptors in angiogenesis, progression and pathophysiology of pituitary tumours is still poorly understood. In the present work, the expression and localization of the angiogenic factor VEGF-A and the lymphangiogenic factor VEGF-C, as well as VEGF receptors (VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1), have been studied in normal and tumoural pituitary tissue and in transformed pituitary tumour cell lines. In addition, the role and mechanism of action of VEGFR-1 ligands have been investigated in normal and transformed rat pituitary cells. Immunohistochemical investigations in 3 normal human adenohypophyses showed that VEGFR-2 and neuropilin-1 were localized in blood vessel endothelial cells, while VEGFR-1 was found in endocrine cells. VEGF-A significantly induced ACTH and prolactin secretion in normal rat pituitary cell cultures, indicating a role of VEGF-A and VEGFR-1 in the regulation of the secretion of these pituitary hormones. In contrast, VEGFR-2 and its co-receptor neuropilin-1 may be needed to maintain optimal intrapituitary vascularization and blood vessel permeability. Although no lymphatic vessels were identified in normal adenohypophysis, the lymphangiogenic factor VEGF-C and its receptor VEGFR-3 were detected by immunohistochemistry, suggesting the involvement of the VEGF-C/VEGFR-3 system, usually implicated in lymphangiogenesis, in the maintenance of blood vessel permeability. The expression of VEGFR-1, VEGFR-2 and neuropilin-1 in a series of 39 pituitary adenomas reflected the same immunohistochemical localization pattern as observed in the normal adenohypophysis tissue. It was highly heterogeneous and mostly no significant correlation with different parameters, such as: tumour type, tumour grade, proliferation index (PI) and blood vessel number, was noticed. Only the absence of VEGFR-2 and neuropilin-1 correlated with a low PI, suggesting a role of these two receptors in increasing vessel permeability and consequently the availability of nutrients and oxygen for tumour cells. Functional studies, with the VEGFR-1-positive somatotrophinoma rat pituitary cell line MtT-S, showed that VEGF-A and the VEGFR-1 specific ligand PlGF, significantly stimulated the cell proliferation, through the activation of PI3K pathway and the induction of the anti-apoptotic factor Bcl-2 and the cell cycle promoter cyclin D1. VEGF-C immunostaining was detected in endocrine tumour cells of 10 adenomas and VEGFR-3 immunopositive vessels were found in 22 tumours, even if only 9 of them were positive for both VEGFR-3 and LYVE-1 (specific lymphatic vessel marker), suggesting that the VEGF-C/VEGFR-3 system may have a role in the regulation of tumour angiogenesis of pituitary adenomas, rather than in lymphangiogenesis, as already shown in other tumour types. In conclusion, the results of the present study provide strong evidence that VEGF may not only have a role in regulating pituitary adenoma neovascularization but also, through VEGFR-1, may affect pituitary adenoma pathophysiology by modulating growth, cell cycle progression and survival of the adenoma cells.