Vascular endothelial growth factor in salivary pleomorphic adenomas: one of the reasons for their poorly vascularized stroma.

To better understand the poorly vascularized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible factor-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ vascular endothelial cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(121) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly vascularized pleomorphic adenomas.