Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features

Abstract

Experimental evidence has shown, both in vitro and in animal models, that neoplastic growth and subsequent metastasis formation depend on the tumor's ability to induce an angiogenic switch. This requires a change in the balance of angiogenic stimulators and inhibitors. To assess the potential role of angiogenesis factors in human thyroid tumor growth and spread, we analyzed their expression by semiquantitative RT-PCR and immunohistochemistry in normal thyroid tissues, benign lesions, and different thyroid carcinomas. Compared to normal tissues, in thyroid neoplasias we observed a consistent increase in vascular endothelial growth factor (VEGF), VEGF-C, and angiopoietin-2 and in their tyrosine kinase receptors KDR, Flt-4, and Tek. In particular, we report the overexpression of angiopoietin-2 and VEGF in thyroid tumor progression from a prevascular to a vascular phase. In fact, we found a strong association between tumor size and high levels of VEGF and angiopoietin-2. Furthermore, our results show an increased expression of VEGF-C in lymph node invasive thyroid tumors and, on the other hand, a decrease of thrombospondin-1, an angioinhibitory factor, in thyroid malignancies capable of hematic spread. These results suggest that, in human thyroid tumors, angiogenesis factors seem involved in neoplastic growth and aggressiveness. Moreover, our findings are in keeping with a recent hypothesis that in the presence of VEGF, angiopoietin-2 may collaborate at the front of invading vascular sprouts, serving as an initial angiogenic signal that accompanies tumor growth.

Figure 1.

Expression of VEGF and its receptors in thyroid tumors. A: Total RNA was extracted from normal thyroid tissues and different thyroid tumor specimens. Semiquantitative RT-PCR analysis was performed using 5 μg of total RNA and intron-spanning primers specific for VEGF, Flt-1, and KDR. Specific oligonucleotides were used to amplify different VEGF isoforms. Four bands were visualized that corresponded to 189aa (460 bp), 165aa (410 bp), 145aa (370 bp), and 121aa (270 bp). The relative intensities of the bands were densitometrically quantified and normalized to the coamplified aldolase signal (Aldo). The numbers at the top of the figure indicate different patients. B: Immunoperoxidase staining of VEGF in normal and neoplastic thyroid tissues. Magnification, ×200. N, normal thyroid; Ade, follicular adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; UTC, undifferentiated thyroid carcinoma; MTC, medullary thyroid carcinoma.

Figure 2.

Up-regulation of angiopoietin 2 in thyroid tumors. Total RNA was extracted from normal thyroid tissues and different thyroid tumors. Semiquantitative RT-PCR analysis was performed using 5 μg of total RNA and intron-spanning primers for angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), and Tek. The relative intensities of the bands were densitometrically quantified and normalized to the coamplified aldolase signal (Aldo). The numbers at the top of the figure indicate different patients. N, normal thyroid; Ade, follicular adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; UTC, undifferentiated thyroid carcinoma; MTC, medullary thyroid carcinoma.

Figure 3.

Expression of VEGF-C and Flt-4 in thyroid tumors. A: Total RNA was extracted from normal thyroid tissue and different thyroid tumor specimens. Semiquantitative RT-PCR analysis was performed using 5 μg of total RNA and intron-spanning primers specific for VEGF-C and Flt-4. The relative intensities of the bands were densitometrically quantified and normalized to the coamplified aldolase signal (Aldo). The numbers at the top of the figure indicate different patients. B: Immunoperoxidase staining of VEGF-C in normal and neoplastic thyroid tissues. Magnification, ×200. N, normal thyroid; Ade, follicular adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; UTC, undifferentiated thyroid carcinoma; MTC, medullary thyroid carcinoma.

Figure 4.

Down-regulation of TSP-1 in thyroid tumors. A: Total RNA was extracted from normal thyroid tissue and different thyroid tumor specimens. Semiquantitative RT-PCR analysis was performed using 5 μg of total RNA and intron-spanning primers specific for TSP-1. The relative intensities of the bands were densitometrically quantified and normalized to the coamplified aldolase signal (Aldo). The numbers at the top of the figure indicate different patients. B: Immunoperoxidase staining of TSP-1 in normal and neoplastic thyroid tissues. Magnification, ×200. N, normal thyroid; Ade, follicular adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; UTC, undifferentiated thyroid carcinoma; MTC, medullary thyroid carcinoma.

Figure 5.

A: Levels of expression of VEGF, VEGF-C, Ang2, and TSP-1 in thyroid papillary microcarcinomas. Semiquantitative RT-PCR analysis was performed as described in Figure 1 ▶ , with specific primers. The relative intensities of the bands were densitometrically quantified and normalized to the coamplified aldolase signal (Aldo). B: Northern blot analysis of mRNAs (5 μg) isolated from normal thyroid tissue and different thyroid tumors. The membrane was analyzed with successive hybridization to VEGF, VEGF-C, Ang2, and β-actin probes. Relative levels of the different transcripts were determined by the phosphorimager instrument and normalized with the β-actin hybridization signal. The numbers at the top of the figure indicate different patients. N, normal thyroid; Ade, follicular adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; UTC, undifferentiated thyroid carcinoma; MTC, medullary thyroid carcinoma.