Colony forming unit endothelial cells do not exhibit telomerase alternative splicing variants and activity

Abstract

Introduction: Endothelial progenitor colony forming unit-endothelial cells (CFU-EC) were first believed to be the progenitors of endothelial cells, named endothelial progenitor cells. Further studies revealed that they are monocytes regulating vasculogenesis. The main hindrance of these cells for therapeutic purposes is their low frequency and limited replicative potentials. This study was undertaken to determine telomerase activity and alternative splicing variants in CFU-EC as a potential cause of limited replicative capacity in these cells.

Methods: CFU-EC were isolated from peripheral blood using a standard cell culture assay. Colonies were detached mechanically and alternative splicing variant mRNA were evaluated using real-time PCR. Telomerase enzyme activity was assessed using telomerase repeat amplification protocol. The same procedures were done on the cancer cell line Calu6 as the positive control.

Results: The cultured peripheral blood mononuclear cells formed colonies with spindle-shaped monocytic cells sprouted from the clusters. These morphological characteristics fulfill the definition of CFU-EC. Telomere length amplification protocol assay revealed no telomerase activity and real-time PCR showed no expression of telomerase enzyme mRNA in CFU-EC. Both parameters were significantly higher in the cancer cell line Calu6 taken as the positive control.

Conclusion: The absence of telomerase activity in the CFU-EC is a result of pre-transcriptional regulation of gene expression rather than other mechanisms for controlling telomerase activity such as post-transcriptional modifications. This finding can explain the limited proliferative activity of CFU-EC cells. We propose that absence of telomerase activity in CFU-EC can be attributable to their more mature monocytic nature that needs further investigations.

Keywords: Telomerase; Endothelial cells; Hemangioblast; Alternative splicing.

Fig. 1

Deletion sites of different variants of hTERT and position of gene specific primers and their sequences. α-deletion site is located on exon 6, while β-deletion site is located on exons7 and 8. Using specific primers, the expression of full-length hTERT and three alternatively spliced variants (α-, ß- and αß-) were analyzed. Sequence of primers used for real-time PCR evaluation of different variants of hTERT expression and β-actin as the house-keeping gene have been shown in the adjacent Table. Exo, exon

Fig. 2

A colony of colony-forming unit endothelial cells.  The cultured peripheral blood mononuclear cells, typically formed colonies with spindle-shaped cells, sprouted from the clusters

Fig. 3

Real-time RT-PCR amplication graph. Low to absent levels of human telomerase reverse transcriptase alternative splicing variant was noted during real-time PCR cycles, despite the high levels of  -actin defined as red and green lines

Fig. 4

Real-time RT-PCR data. The analysis of RT-PCR data showed no expression of human telomerase reverse transcriptase alternative splicing variants in colony forming unit-endothelial cells. The α-actin mRNA, taken as the internal control, was significantly amplified