Transfection of the Human Heme Oxygenase Gene into Rabbit Coronary Microvessel Endothelial Cells
Heme oxygenase* (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents which may induce oxidative injury such as metals, endotoxin, heme-hemoglobin and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has anti-oxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited a ≈3.0-fold increase in enzyme activity and expressed a several-fold induction of human HO mRNA as compared to endogenous rabbit HO mRNA. Transfected and non-transfected cells expressed Factor VIII antigen and exhibited similar acetylated low density lipoprotein uptake (two important features which characterize endothelial cells) with greater than 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin (rHb) and heme as compared to non-transfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.
KeywordsFactor Viii Heme Oxygenase Cationic Liposome Free Heme Heme Oxygenase Activity
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