Subject(s)
Cell Division , Hepatocytes/metabolism , Retroviridae/genetics , Animals , Apoptosis , Cell Transplantation , Gene Expression , Gene Transfer, Horizontal , Genetic Vectors , Green Fluorescent Proteins , Hepatocytes/cytology , Humans , Kinetics , Luminescent Proteins/genetics , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , beta-Galactosidase/geneticsABSTRACT
Stable gene transfer into hepatocytes has been proposed to compensate for genetic deficiencies that affect liver function, or to deliver diffusible factors into the circulation. This strategy can be achieved using retroviral vectors; however, cell division must occur. We describe a simple and reproductive method that enables the induction of hepatocyte replication in a controlled fashion, thus allowing an efficient in vivo retroviral liver transduction that is applicable to mouse models of human genetic disorders. The approach is based on liver susceptibility to apoptosis via the Fas/CD95 pathway. We show that, 4 days following a single Fas agonist antibody (JO2) injection, hepatocyte replication occurs, the intensity of which is correlated with the level of the induced hepatic cytolysis. This treatment enables in vivo liver transduction, and its efficiency also correlates with the level of hepatic cytolysis. When recombinant retroviral vectors were infused intravenously during the period of hepatocyte replication, 15.4% +/- 1.7% of the hepatocytes were transduced, reaching up to 32.5%.
Subject(s)
Gene Transfer Techniques , Genetic Vectors , Liver Regeneration/physiology , Retroviridae/genetics , fas Receptor/physiology , Alanine Transaminase/blood , Animals , Antibodies, Monoclonal/pharmacology , Cell Division/drug effects , Dose-Response Relationship, Drug , Female , Hepatocytes/cytology , Injections , Mice , Mice, Inbred C57BL , Transduction, Genetic , Ultrafiltration , fas Receptor/immunologyABSTRACT
BACKGROUND: Apoptosis has been shown to contribute to myocardial reperfusion injury. It has been suggested that, in reducing the apoptotic component within the ischemic area at risk, Bcl-2 overexpression could lead to a ventricular function improvement. METHODS: Transgenic mice overexpressing the anti-apoptotic human Bcl-2 cDNA in heart were subjected to a 1-h left coronary artery occlusion followed by a 24-h reperfusion. At the end of the experiment, left ventricular function was assessed by two-dimensional echocardiography. After sacrifice, the area at risk (AR) and the infarct area (IA) were determined by Evans blue and triphenyltetrazolium chloride staining, respectively. The extent of apoptosis was assessed by the TUNEL method. Non-transgenic littermates served as controls. RESULTS: Baseline AR was not different between Bcl-2 transgenic mice and their wild-type littermates. In contrast, left ventricular ejection fraction was significantly improved in the transgenic mice line (61.25 +/- 4.0%) compared to non-transgenic littermates (43.2 +/- 5.0%, p < 0.01). This functional amelioration was correlated with a significant reduction of infarct size in transgenic animals (IA/AR 18.51 +/- 3.4% vs 50.83 +/- 8.4% in non-transgenic littermates). Finally, apoptotic nuclei were less numerous in transgenic mice than in controls as quantified by TUNEL analysis (8.1 +/- 2.2% vs 20.6 +/- 4.4%). CONCLUSIONS: Bcl-2 overexpression is effective in reducing myocardial reperfusion injury and improving heart function. This benefit correlates with a reduction of cardiomyocyte apoptosis. The apoptotic component of ischemia/reperfusion injury could therefore constitute a new therapeutic target in the acute phase of myocardial infarction.
