Potential proliferative effect of lipopolysaccharide preconditioning on human umbilical cord blood-derived endothelial progenitor cells
Endothelial progenitors cells (EPCs) are important for the development of cell therapies for various diseases. However, the major obstacles in developing such therapies are low quantities of EPCs that can be generated from the patient and the lack of adequate non-invasive imaging approach for in vivo monitoring of transplanted cells. Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with gram-negative bacterial infections. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of EPCs. The objective of this study was to determine the ability of human umbilical cord blood (hUCB) EPCs to be isolated and identified in vitro and examine the effect of LPS on EPCs- TLR4 signalling pathway. 5 hUCB samples were collected for EPCs isolation and cultured on fibronectin coated wells in endothelial media supplement. Their phenotypes were confirmed by uptake of acetylated LDL and binding of fluorescein isothiocyanate (FITC) - labeled Ulex europaeus agglutinin 1 (UEA-1) lectin. LPS was added to EPCs culture by different concentrations (0.1, 0.5 and 1 µg/ml) to assess its effect on EPCs proliferation. The following genes; human vascular endothelial growth factor receptor-2 (hVEGFR-2), TLR4, vascular endothelial-cadherin (VE-Cadherin), endothelial nitric oxide synthase (eNOS) and heme oxygenase (HO-1) from the culture EPCs (control group) and EPCs preconditioned with 0.1 µg/ml LPS (preconditioned treated group) were also assessed by Real-time qPCR. EPCs cultured from hUCB were isolated, identified and highly significant proliferated in response to 0.1 µg/ml LPS compared to other concentrations. The expression of hVEGFR-2, TLR4, VE-Cadherin, eNOS and HO-1 by EPCS preconditioned with LPS compared to EPCs was significantly increased in response to the toll-like receptor agonist LPS. hUCB is highly valuable rich source for EPCs. Data of Quantitative genes expression indicate that LPS recognition by EPCs-TLR4 activates the corresponding signal pathway.
Key words: Human umbilical cord blood (hUCB), Endothelial progenitors cells (EPCs), lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4).
Abbreviation: EPCs, Endothelial progenitors cells; LPS, lipopolysaccharide; hUCB, human umbilical cord blood; FITC, fluorescein isothiocyanate; TLR4, toll-like receptor 4; hVEGFR-2, human vascular endothelial growth factor receptor-2; TLR4, VE-Cadherin, vascular endothelial-cadherin; eNOS, endothelial nitric oxide synthase; HO-1, heme oxygenase; LDL, low-density lipoprotein.