Enhanced expression of the tie receptor tyrosine kinase mesenger RNA in the vascular endothelium of metastatic melanomas.

Angiogenesis of human melanomas has been the focus of intense interest since it was shown that the spread and prognosis of primary tumors is correlated with their vascularization (N. Weidner, J. P. Semple, W. R. Welch, and J. Folkman, N. Engl. J. Med., 324: 1-8, 1991). Basic fibroblast growth factor (bFGF) and its high-affinity receptor FGFR-1 have been implicated in melanoma growth and angiogenesis (R. Halaban, Y. Funasaka, J. Lee, J. Rubin, D. Ron, and D. Birnbaum, Fibroblast Growth Factors in Normal and Malignant Melanocytes, pp. 232-243. New York: The New York Academy of Sciences, 1991). We have studied the expression of the Tie endothelial cell receptor tyrosine kinase mRNA in skin and primary cutaneous melanomas as well as in their skin and brain metastases by in situ hybridization. The Tie probe hybridized very weakly with the vascular endothelium of capillaries of normal skin, while it was detected in larger arteries and veins as well as in capillaries around sweat glands. However, capillaries and medium-sized vessels within cutaneous and brain metastases of melanoma were strongly positive for Tie mRNA. In contrast, endothelial cells contained very little or no FGFR-1 transcripts, whereas abundant FGFR-1 mRNA was present in melanoma tumor cells and in fibrovascular stroma. In agreement with these findings, a Tie-specific amplified cDNA band was obtained by reverse transcription-polymerase chain reaction from melanoma metastases but not from normal skin. These results suggest a role for the Tie receptor in the angiogenesis associated with melanoma metastases.

Tie endothelial cell-specific receptor tyrosine kinase is upregulated in the vasculature of arteriovenous malformations.

Arteriovenous malformations (AVMs) are congenital lesions composed of abnormal vasculature, with no capillary component, and are clinically significant due to their tendency to spontaneously hemorrhage. The mechanisms regulating the genesis and progression of these lesions are unknown. In order to study the role of angiogenesis in AVMs, we have analyzed the expression of the endothelial cell mitogen vascular endothelial growth factor (VEGF) and a novel endothelial cell-specific receptor tyrosine kinase, Tie, by in situ hybridization and immunohistochemistry in these malformations and surrounding brain tissue. We have previously shown upregulation of Tie accompanying wound healing and tumor progression. In this study, we demonstrate significantly elevated levels of Tie mRNA and protein in AVM and surrounding brain vasculature. Upregulation of VEGF mRNA was observed in the cells of brain parenchyma adjacent to the AVM, and VEGF protein was detected in this tissue as well as in AVM endothelia. Normal brain, in comparison, expressed little or no Tie or VEGF. The significant upregulation of VEGF and Tie in AVMs may indicate some ongoing angiogenesis, possibly contributing to the slow growth and maintenance of the AVM, and could be of potential use in the therapeutic targeting of these lesions.

Expression of growth factors and growth factor receptors in capillary hemangioblastoma.

To elucidate the mechanisms underlying the regulation of growth and differentiation of capillary hemangioblastoma we studied the expression of selected growth factors and growth factor receptors by immunocytochemistry. As stromal cells of capillary hemangioblastoma express high levels of vascular endothelial growth factor (VEGF) and placental growth factor (P1GF) mRNA, we studied the distribution of the corresponding VEGF and P1GF proteins. We also studied the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptors (PDGFR) because their ligands have been reported to promote angiogenesis. The stromal cells expressed abundant EGFR and, in addition, some stromal cells expressed PDGFR-alpha but not PDGFR-beta. In contrast, the endothelial cells co-expressed PDGFR-alpha and PDGFR-beta. VEGF and P1GF were expressed by scattered stromal cells; however, more intense staining was observed in the endothelial cells of the intratumoral blood vessels, possibly indicating the secreted growth factors bound to their target receptors. We conclude that capillary hemangioblastomas express a variety of growth factor receptors and ligands, potentially involved in both autocrine and paracrine loops. The uniformly high EGFR expression is unique among brain tumors and may be associated with the typical morphology of capillary hemangioblastoma. The expression of highly angiogenic growth factors and their receptors may contribute to the rich vascularity of this enigmatic tumor.

Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis.

Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.

Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors.

Expression of vascular endothelial growth factor and placenta growth factor in human placenta.

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.

Expression of Mad, an antagonist of Myc oncoprotein function, in differentiating keratinocytes during tumorigenesis of the skin.

The Myc oncoprotein is associated with cell proliferation and is often down-regulated during cell differentiation. The related Mad transcription factor, which antagonises Myc activity, is highly expressed in epidermal keratinocytes. Mad also inhibits cell proliferation in vitro. To study Mad expression in keratinocyte proliferation and differentiation, we have analysed Mad RNA expression in regenerating and hyperproliferative epidermal lesions and epidermal tumours of varying degrees of differentiation using the RNA in situ hybridisation and RNAase protection techniques. Mad was strongly expressed in differentiating suprabasal keratinocytes in healing dermal wounds and in benign hyperproliferative conditions, but also in squamous cell carcinomas, in which the keratinocytes retain their differentiation potential. However, Mad expression was lost in palisading basal carcinoma cells and poorly differentiated squamous cell carcinomas, which lacked the epithelial differentiation marker syndecan-1. We therefore suggest that Mad expression is closely associated with epithelial cell differentiation, and that this association is retained in epithelial tumours of the skin.