Abstract
Vascular homeostasis and regeneration in ischemic tissue relies on intrinsic competence of the tissue to rapidly recruit endothelial cells for vascularization. The mononuclear cell (MNC) fraction of blood contains circulating progenitors committed to endothelial lineage. These progenitors give rise to endothelial colony-forming cells (ECFCs) that actively participate in neovascularization of ischemic tissue. To evaluate if the initial clonal outgrowth of ECFCs from cord (CB) and peripheral blood (PB) was stimulated by hypoxic conditions, MNCs obtained from CB and PB were subjected to 20 and 1% O2 cell culture conditions. Clonal outgrowth was followed during a 30 day incubation period. Hypoxia impaired the initial outgrowth of ECFC colonies from CB and also reduced their number that were developing from PB MNCs. Three days of oxygenation (20% O2) prior to hypoxia could overcome the initial CB-ECFC outgrowth. Once proliferating and subcultured the CB-ECFCs growth was only modestly affected by hypoxia; proliferation of PB-ECFCs was reduced to a similar extent (18–30% reduction). Early passages of subcultured CB- and PB-ECFCs contained only viable cells and few if any senescent cells. Tube formation by subcultured PB-ECFCs was also markedly inhibited by continuous exposure to 1% O2. Gene expression profiles point to regulation of the cell cycle and metabolism as major altered gene clusters. Finally we discuss our counterintuitive observations in the context of the important role that hypoxia has in promoting neovascularization.
Highlights
The majority of wounds heal through physiological tissue repair
Subsequent studies showed that the cells originally identified as endothelial progenitor cells harbored various cell types, in particular myeloid cells that acquired endothelial marker properties and endothelial colony-forming cells (ECFCs), that actively participate in neovascularization [9,10,11,12,13]
To investigate whether hypoxia stimulates the initial outgrowth of ECFCs from progenitor cells that reside within the mononuclear cell (MNC) fractions of cord- and peripheral blood, CB-MNC inoculates of 14 individual donors were each incubated at 1% or 20% O2 for up to 30 days with regular renewal of the culture medium
Summary
The majority of wounds heal through physiological tissue repair. But if tissue repair fails, tissue engineering or regenerative medicine and transplantation is necessary [1]. One of the problems during regenerative medicine is that oxygen diffusion is limited in a cellular tissue-engineered scaffold [2], resulting in reduced (hypoxia) or lack of oxygen (anoxia) within the deeper regions of the scaffold and cell death [2, 3]. In many disease conditions or after implantation of an engineered graft the ability of the endothelium to generate new vessels proceeds too slowly to overcome tissue hypoxia and subsequent cell death. ECFCs— called blood-originated endothelial cells (BOECs)—exhibit high proliferative and colony-forming ability, do belong to the endothelial cell lineage and not to the hematopoietic cell lineage, and possess robust in vitro and in vivo neovascularization ability including participation in the lining of new vessels [9, 14]
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