Angiogenesis and vascular architecture in pheochromocytomas: distinctive traits in malignant tumors

Abstract

Angiogenesis is a critical step in tumor growth and metastatic invasion. We here report the study of the vascular status of 10 benign and 9 malignant pheochromocytomas. We examined the vascular architecture after immunostaining endothelial cells (CD34) and vascular smooth muscle cells (alpha-actin) and identified a vascular pattern characteristic of malignant lesions. To define a gene expression profile indicative of the invasive phenotype, we studied by in situ hybridization the expression of genes encoding several pro- and anti-angiogenic factors [hypoxia-inducible factor (HIF-1 alpha), EPAS1, vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins and their receptor Tie2, five genes of the endothelin system, and thrombospondin 1]. A semiquantitative evaluation of the labeling revealed an induction of genes encoding EPAS1, VEGF, VEGFR-1, VEGFR-2, endothelin receptor, type B (ETB) and endothelin receptor, type A (ETA) in malignant pheochromocytomas as compared to benign tumors. These differences were observed in tumor cells, in endothelial cells, or in both. Quantification by real-time reverse-transcriptase polymerase chain reaction showed an increase of EPAS1, VEGF, and ETB transcripts of 4.5-, 3.5-, and 10-fold, respectively, in malignant versus benign tumors. Furthermore, we observed a strong correlation between the expression of EPAS1 and VEGF in tumoral tissue and between EPAS1 and ETB in endothelial cells. Altogether, our observations show that analysis of angiogenesis provides promising new criteria for the diagnosis of malignant pheochromocytomas.

Figures

Figure 1.

Immunostaining of chromaffin cells. Immunolabeling of tyrosine hydroxylase (A, C) or neuron-specific enolase (B, D) reveals the nodules of tumoral cells of a benign (A, B) and a malignant (C, D) pheochromocytoma. No apparent difference in the signal intensity between benign and malignant tumors was observed. Scale bar, 100 μm.

Figure 2.

Vascular architecture. The vascular network of a normal adrenal medulla (A), and a benign (B) and four malignant (C–F) pheochromocytomas is observed under light microscopy. Sections were immunostained for ECs with an anti-CD34 antibody and for vascular smooth muscle cells with an anti-α-actin antibody (insets). These structures illustrate archetypal examples of the regular (B) and the irregular vascular patterns (C–F). Note the presence of vascular entities forming arcs (C) as well as large (D) or tight (E) networks. Images in which straight blood vessels run in parallel have also been observed (F). In the irregular patterns, the vascular density is inferior to that observed in the regular pattern, as illustrated by the presence of avascular tumor nodules (D). Scale bars, 200 μm.

Figure 3.

Sites of expression of angiogenesis-related factors in pheochromocytomas revealed by observation under bright-field illumination at a high magnification. VEGFR-1, VEGFR-2, ETB, Tie2, TSP1, and PPET-1 are restricted to ECs. Note the presence of PPET-1 in very few ECs in the blood vessel wall (arrows). EPAS1 is present in both ECs and chromaffin cells, which express large amounts of VEGF transcript. Genes encoding HIF-1α and PPET-3 are ubiquitously expressed whereas ETA has a heterogeneous pattern of expression. It is present either in pericytes (left) or in tumor cells (right). Ang-2 is not detectable in these tissues. Scale bar, 100 μm.

Figure 4.

Expression of EPAS1 transcripts in a benign (A, B) and a malignant (C, D) pheochromocytoma. Observation under dark-field (A, C) or bright-field (B, D) illuminations. Note the overexpression of EPAS1 in the malignant tumor, especially in ECs of the blood vessels wall (arrows). Scale bars, 100 μm.

Figure 5.

In situ hybridization of genes of the VEGF system. Serial sections of a benign (A–C) and a malignant (D–F) pheochromocytoma radiolabeled with VEGF (A, D), VEGFR-1 (B, E), and VEGFR-2 (C, F) probes. Signals are observed under dark-field illumination. The presence of VEGF transcripts is detected in tumoral cells, whereas the receptors are expressed in vascular ECs. Note that these three markers are expressed at a higher level in the malignant tissue. Scale bar, 100 μm.

Figure 6.

Expression of the ETB receptor in a benign (A, B) and a malignant (C, D) pheochromocytoma. Observation under dark-field illumination (A, C) reveals a higher density of ETB-positive ECs in the malignant tissue (C) compared to the benign neoplasm (A). The intensity of the signal for one cell, as shown in bright-field observations (B, D) is also stronger in the malignant pheochromocytoma (D). Arrows, blood vessels. Scale bars, 100 μm.

Figure 7.

Expression of the ETA receptor in a benign (A) and two malignant (B, C) pheochromocytomas. No ETA labeling is detected in a benign neoplasm whereas there is a strong signal in malignant tissues, either in pericytes (B) or in tumor cells (C). Dark-field pictures. Arrows, blood vessels. Scale bar, 100 μm.

Figure 8.

Expression levels of endothelin receptors in benign and malignant pheochromocytomas: comparison of individual values. The values correspond to the intensity of labeling in ECs for ETB gene (A) and in either pericytes or tumor cells for ETA mRNA (B). Note the absence of overlap between these levels of expression in patients with benign tumors when compared to the malignant pheochromocytomas.

Figure 9.

Real-time RT-PCR quantification of EPAS1, VEGF, and ETB transcripts in benign versus malignant pheochromocytomas. Mean values ± SEM of relative mRNA expressions in two benign (hatched bars) versus two malignant (black bars) tumors are presented as a gene/18S ratio.