Gene expression profiling in an in vitro model of angiogenesis

Abstract

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Figure 1.

TaqMan analysis of the changes in gene expression of 26 genes identified by the GeneCalling analysis over the time period of 30 minutes to 46.5 hours. The changes in gene expression were grouped into four patterns, shown as Group 1 (rapid elevation in mRNA, peaking at 2–4 hours, then declining to baseline levels by 24 hours), Group II (more delayed elevation in mRNA, peaking at 8–12 hours, then declining to near-baseline levels by 46.5 hours), Group III (mRNA levels rising somewhat later than Group I or II, peaking at 12–46.5 hours, and remaining markedly above baseline levels at 46.5 hours), and Group IV (mRNA levels declining from the initial value observed at 30 minutes, and by 16–24 hours mRNA levels were below those obtained from quiescent HUVECs (see Materials and Methods)).

Figure 2.

In situ hybridization demonstrating expression of genes identified from the differential expression analysis in the vasculature associated with tumors and with inflammatory disease. A–D: Hematoxylin-eosin (A and C) and in situ (B and D) hybridization demonstrating vascular expression (arrows) of stanniocalcin precursor mRNA in squamous cell carcinoma (A and B) and ductal mammary adenocarcinoma (C and D). E and F: Hematoxylin and eosin stain (E) and in situ (F) hybridization of osteonidogen mRNA in an arteriole (arrows) of inflamed appendix. G and H: Hematoxylin and eosin stain (G) and in situ (H) hybridization of podocalyxin expression in vessels surrounding lung squamous cell carcinoma (arrows). I and J: Hematoxylin and eosin stain (I) and in situ (J) hybridization of ADAMTS-4 expression adjacent to chondrosarcoma.