Gene transfer into rat heart-derived endothelial cells1

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Abstract

Objective:

Progressive graft arteriosclerosis is responsible for the majority of late deaths in cardiac transplant recipients. Despite many investigations, the pathogenesis of this disease remains undetermined and its control inadequate. A somatic gene transfer during the cold ischemic time and thus before transplantation might be a new therapeutic tool. This approach allows a long incubation time of the DNA and a safe transfer with liposomes and transferrin with less adverse effects for the organ recipient.

Methods:

The target cells (microvascular endothelial cells (MVECs)) for this gene transfer were isolated from rat hearts by perfusion with collagenase via an aortic cannulae. The cells were purified by changing the medium 30 min after subcultivation in order to remove fibroblasts and smooth muscle cells. The endothelial cells (ECs) were identified by typical morphology and the uptake of Dil-Ac-LDL. The gene transfer was carried out with a β-galactosidase reporter plasmid (pCMVβ), cationic liposomes (Lipofectin®), and transferrin. Different transfection solutions were prepared with or without serum, and with different plasmid-liposome ratios and transferrin concentrations. The transfer rate was monitored with a semiquantitative orthonitrophenyl-β-d-galactoside (ONPG) assay and histologically by X-Gal staining. The cytotoxicity of this procedure was determined with a colorimetric ELISA with Alamar blue®. The cardioplegic property of the transfection solution was tested in a Langendorff perfusion system monitoring the coronary blood flow over time after a cold ischemic time of 4 h.

Results:

The maximal gene expression could be detected after transfection with 4 μl Lipofectin, 2 μg pCMVβ, and 16 μg transferrin/200 μl transfection solution. Under these conditions 60% of the cells showed a blue staining with X-Gal. Only 20% of the cells died during transfection. The lowest cytotoxicity during cold ischemic time for ECs was assessed with normal cell culture medium and the Buckberg solution. The best coronary flow rates after 4 h cold ischemia of the heart were measured for cardioplegia with St. Thomas and Buckberg solutions. In summary, the best transfection solution with a good cardioplegic property was the Buckberg solution.

Conclusions:

Finally, the results of this study show that an effective DNA delivery with a low toxicity into ECs is possible with a combination of liposomes and transferrin. This method might be useful for a safe and effective gene transfer into solid organs during the cold ischemic time and thus a therapeutic tool for chronic rejection.

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