Isolation and characterization of human endothelial colony forming cells (ECFCs) and effect of oxidative stress on their differentiation

PhD ThesisEndothelial dysfunction plays a pivotal role in the development and progression of atherosclerosis. Atherogenic risk factors harbor the potential to injure the endothelial layer by promoting apoptosis, preceded by an inflammatory reaction in the vessel wall. The maintenance of the endothelial layer is therefore crucial to ensure its integrity. More recent studies suggest an important role of endothelial progenitor cells (EPCs) in contributing towards reendothelialization after vascular injury. These EPCs home to sites of hypoxia or ischemia and take part in vascular repair. Recent studies have shown that a rare population of EPCs called endothelial colony forming cells or ECFCs can be derived from peripheral and cord blood and can be transplanted into immunodeficient mice that have demonstrated the ability to form chimeric blood vessels. Hyperoxia has been shown to be a model of mild oxidative stress, and this oxidative stress is characterized by an increase in reactive oxygen species (ROS) levels that can lead to a wide range of chemical reactions resulting in cellular necrosis and apoptosis through lipid peroxidation and DNA damage. We therefore proposed that by growing endothelial cells in vitro in hyperoxic conditions can in turn lead to cellular damage and activation of pro inflammatory markers in these cells. PGC-1α belongs to a small family of transcriptional coactivators which have been shown to regulate reactive oxygen species generation and apoptosis in mature endothelial cells. The mechanisms through which PGC-1α activates gene expression are poorly understood. This study aims at identifying a molecular signature of human endothelial progenitor cells (ECFCs) so that they can be distinguished from mature endothelial cells (HUVEC) and identifying the role of PGC-1α and its downstream targets when these endothelial progenitors and mature endothelial cells are exposed to oxidative stress. In order to achieve that goal we isolated endothelial colony forming cells and mature endothelial cells from the same donor. This was followed by their molecular profiling at mRNA level using qPCR and at protein level using immunofluorescence and FACS analysis. Cells were grown till late passages to study the growth kinetics of ECFCs and HUVEC in normoxia and hyperoxia. Telomerase activity was measured using TRAP assay and telomere length measurement was done using flow FISH. Low density array was carried out in order to distinguish between ECFCs and HUVEC in terms of gene expression analysis. The results showed that ECFCs were distinguished from early EPCs and HUVEC. PGC-1 alpha levels were up regulated in ECFCs when exposed to hyperoxia and this in turn activated several other genes that are involved in angiogenesis, oxidative phosphorylation and electron transport chain. ECFCs and HUVEC have shown to behave differently under conditions of oxidative stress and we found ECFCs to be more resistant to stress than HUVEC as shown in their growth kinetics