Abstract
BackgroundMonocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.ResultsHuman blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.ConclusionsThis report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.
Highlights
Monocyte-derived macrophages contribute to atherosclerotic plaque formation
On day 8, all cultures transduced with VRX1090 showed enhanced green fluorescent protein (EGFP)-positive cells by microscopic examination (Figure 1A)
Flow cytometric analysis carried out on day 9 showed a peak shift in data histograms corresponding to the presence of EGFP-positive cells for all transduced cultures compared with non-transduced control cells (Figure 1A)
Summary
Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Coronary artery disease develops as a consequence of lipid deposition and foam cell formation in the arterial wall leading to the development of atherosclerotic plaques [2]. Macrophages are present in virtually every atherosclerotic plaque and have an important role in foam cell and Circulating blood monocytes differentiate into macrophages. Macrophages produce proinflammatory and proangiogenic mediators, and function as host cells for pathogens. Due to these functions, macrophages can contribute to the initiation and progression of a wide variety of diseases. Macrophages can contribute to the initiation and progression of a wide variety of diseases Directly manipulating these hematopoietic cell subsets could have significant therapeutic value [5]
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