George Boguslawski

High-efficiency transient transfection of endothelial cells for functional analysis

The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence‐activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5‐fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1‐phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl‐terminal domain of the β‐adrenergic receptor kinase (βARKct), which inhibits signaling through the βγ dimer of heterotrimeric G‐proteins. SPP‐induced chemotaxis in sorted cells coexpressing βARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a βγ‐dependent pathway. However, no significant inhibition was observed in cells transfected with βARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.—Kovala, A. T., Harvey, K. A., McGlynn, P., Boguslawski, G., Garcia, J. G. N., English, D. High‐efficiency transient transfection of endothelial cells for functional analysis. The FASEB J. 14, 2486–2494 (2000)

Continuously-infused human C-reactive protein is neither proatherosclerotic nor proinflammatory in apolipoprotein E-deficient mice.

Studies of human native C-reactive protein (nCRP) in mice have shown effects ranging from proatherogenic, to antiatherogenic, to no effect. It is likely that these disparities are related to (a) the use, in some studies, of contaminated nCRP, or to (b) variation in CRP levels associated with either its episodic administration or the use of CRP-transgenic mice. In our study, 12-week-old male apolipoprotein E–deficient (apoE −/−) mice, maintained on a Western diet, received azide- and endotoxin-free nCRP (n = 23) or placebo (n = 23) continuously via osmotic pumps (20.4 μg/day) for 4 weeks. CRP-treated and control mice developed similar atherosclerotic lesions in whole aortas (nCRP: 10.4 ± 4.7% vs. controls: 11.7 ± 4.4%, P = 0.76) and aortic roots (nCRP: 65.0 ± 7.8% vs. controls: 64.7 ± 9.7%, P = 0.94). No differences were observed in macrophage or T-lymphocyte infiltrates and there was no meaningful change in VCAM-1 or IL-6 expression, in the levels of soluble VCAM-1, or in circulating proinflammatory (IL-1β, IL-6, IL-12p40, IL-12p70, TNF-α, and INF-γ), or anti-inflammatory (IL-4 and IL-10) cytokines. We conclude that continuous infusion of uncontaminated nCRP in apoE −/− mice is not associated with increased atherosclerosis, does not alter systemic or local inflammation, and does not affect endothelial activation. These observations suggest that alternative approaches to study CRP (perhaps using different pentraxins in the mouse model or using a rabbit model instead of a mouse model) are needed to evaluate the effects of pentraxins on atherosclerosis.