Question

If you were to have completed the experiment using the Tube Formation Assay technique, what would have been some of the expected results and what would have been some of the potential difficulties you might have encountered during the protocol?

Answer 1

Part-1

Gene Expression Results during Early Tubulogenesis-

Endothelial cell tube formation takes almost 6–12 h for completion (Fig. 1)⇓ . Inclusion of the transcriptional inhibitor actinomycin D significantly but incompletely inhibited tubule network formation, but only when added within the first hour of culture (P ≤ 0.05, Student’s t test; Prism Software, San Diego, CA), indicating a contribution of new gene expression for HUVEC differentiation into tubes. No effect of transcriptional blockade with actinomycin D on extent or complexity of the tubules formed was observed when actinomycin D was added to and maintained in culture after the first 60-min period. Nylon cDNA microarrays were used to evaluate gene expression changes during the first hour of incubation of HUVECs on Matrigel. Parallel arrays were hybridized with probes made from aliquots of the same control mRNA for intrasample variability and from different RNA isolations for intersample variability. Similarly, different lots of arrays were tested. P-SCAN software was used to generate scatter plots of expression intensities. Intrasample variability studies indicated that 100 of the 3200 points expressed above membrane background were observed at a ≥3-fold change in gene expression on only one of the paired arrays and were false positive (false positive rate, 3.1%). At a 5-fold level of stringency, fewer points were differentially expressed between the controls, yielding a false positive rate of only 0.4% of expressed genes. Control samples from two independent tubulogenesis assays were used to probe microarrays from the same lot for the intersample variability studies, showing 17 (2%) and 3 (0.3%) false positives at 3- and 5-fold stringency, respectively. We therefore chose to operate at the 5-fold level of stringency of differential expression.

By these criteria, 31 transcripts were expressed differentially (false positive rate, 0.4% of genes queried) within the first hour after attachment . Multiple gene classes were represented, including transcription, translation regulation, cell structure, and cell adhesion. The expression of caldesmon, a cytoskeletal protein that binds to actin, myosin, tropomyosin, and calmodulin not previously linked to endothelial cell function or differentiation was markedly and reproducibly down-regulated RT-PCR analysis reproducibly confirmed this reduction in gene expression (12-fold). Caldesmon protein expression was investigated in the differentiating endothelial cells by use of laser capture microdissection of HUVEC tubules followed by immunoblot analysis. Microdissection of single attached cells provided the protein controls; HUVEC tubules and network nodal points were microdissected for the differentiated cell lysates.

This approach essentially allowed us to do a subtractive analysis of gene expression occurring during the first hour of tube formation.

The result of the experiment is, as you didn't have mentioned clearly the field in which experiment the tube formation technique is used,the particular data based results can not be given accurately.

Part-2

Potential difficulties-

There are some disadvantages also to conduct the endothelial cell tube formation assay.

1.One major problem is that, because there are different types of endothelial cells and support matrices, the results of the assay can vary greatly depending on which type of cell and matrix is used. 2.The endothelial cells used (HUVECs, HAECs or HMVECs) are primary cells, they are costly to get and have variability compared to immortalized cells. The primary cells have limited passages for use, and are therefore not suitable for long term angiogenesis experiments. For reliable data, one should always use the same types for the assay.

3.As with other in vitro assays, the results of a tube formation assay should be confirmed in vivo, because results from the controlled and artificial conditions of two-dimensional tissue culture may not always be reflected in the complex biosystem of a living organism.

4.It is also important to keep in mind that this type of assay can only be used to demonstrate endothelial cell tube formation, and should not be used to test other, non-endothelial, tube forming cells.