Genomic approach to identify factors that drive the formation of three-dimensional structures by EA.hy926 endothelial cells

Abstract

Understanding the mechanisms responsible for tube formation by endothelial cells (ECs) is of major interest and importance in medicine and tissue engineering. Endothelial cells of the human cell line EA.hy926 behave ambivalently when cultured on a random positioning machine (RPM) simulating microgravity. Some cells form tube-like three-dimensional (3D) aggregates, while other cells (AD) continue to grow adherently. Between the fifth and seventh day of culturing, the two types of cell growth achieve the greatest balance. We harvested ECs that grew either adherently or as 3D aggregates separately after five and seven days of incubation on the RPM, and applied gene array analysis and PCR techniques to investigate their gene expression profiles in comparison to ECs growing adherently under normal static 1 g laboratory conditions for equal periods of time. Using gene arrays, 1,625 differentially expressed genes were identified. A strong overrepresentation of transient expression differences was found in the five-day, RPM-treated samples, where the number of genes being differentially expressed in comparison to 1 g cells was highest as well as the degree of alteration regarding distinct genes. We found 27 genes whose levels of expression were changed at least 4-fold in RPM-treated cells as compared to 1 g controls. These genes code for signal transduction and angiogenic factors, cell adhesion, membrane transport proteins or enzymes involved in serine biosynthesis. Fifteen of them, with IL8 (interleukin 8) and VWF (von Willebrand factor) the most prominently affected, showed linkages to genes of another 20 proteins that are important in cell structure maintenance and angiogenesis and extended their network of interaction. Thus, the study reveals numerous genes, which mutually influence each other during initiation of 3D growth of endothelial cells.

Figure 1. Immunocytochemistry.

(A) Laminin-immunocytochemistry of EA.hy926 cells cultured for five days on the RPM. (B) Laminin-immunocytochemistry of EA.hy926 cells cultured for five days in a normal laboratory incubator.

Figure 2. Microarray Analysis.

(A) The first two PCs of the PCA of 31,991 expressed transcripts describe 32.1% of the dataset variance. The five-day RPM samples showed a separation from 1 g controls and seven-day RPM samples in PC #1 (22.5% of the variance). (B) K-mean clustering of the 1625 significantly regulated probes. The color scale represents upregulation (red) or downregulation (blue) of probes. Outliers are marked in orange (upregulated) or green (downregulated). (C–H) STRING visualization of known and predicted physical and functional interactions between the proteins organized in cluster 1 (C), clusters 2, 7 and 9 (D), clusters 3 and 5 (E), cluster 4 (F), cluster 6 (G) and cluster 8 (H).

Figure 3. Interaction Analysis.

STRING visualization of interactions between the proteins of genes playing a role in 3D aggregation and shown in Table 3 (surrounded by a red line) and genes whose level of interaction was changed at least 4-fold (Table 2). IL-8 belongs to both groups.